Assays for compounds that modulate or alter cyclin E activity

ABSTRACT

Whole cell or cell-free test systems for screening and identifying compounds that modulate or alter Cyclin E activity. Whole cell assays measure the G1 phase of cells in the presence or absence of the test compound. Additional whole cell or cell-free assays measure binding of cyclin E to a cell division kinase, measure cyclin E activity directly, or measure the cell division kinase activity directly in the presence or absence of the test compound.

This invention was made with government support under grant CA48718awarded by the National Institutes of Health. The government has certainrights in the invention.

This application is a continuation of Ser. No. 07/947,311, filed Sep.16, 1992, now U.S. Pat. No. 5,449,755, which is a continuation-in-partof Ser. No. 07/764,309, filed Sep. 20, 1991, now abandoned.

FIELD OF THE INVENTION

This invention relates to genetic engineering involving recombinant DNAtechnology, and particularly to the identification of a nucleotidesequence encoding human cyclin E that controls the rate of cell growthby controlling progression at G1 phase of the cell cycle and entry intothe S phase.

BACKGROUND OF THE INVENTION

A major goal in studying the growth and differentiation of highereukaryotic cells is to describe in biochemical terms the pathways,enzymes, and cofactors that regulate progression through the cell cycle,and in particular through the transitions from G1 phase into S phase,and from G2 phase into M phase. Proteins, now known as cyclins, weredescribed in fertilized sea urchin and clam eggs as members of a smallnumber of proteins whose synthesis was greatly stimulated followingfertilization (in the appended Citations: Evans, et. al., 1983) andwhose levels decreased at each mitosis. Cyclin A (Swenson et al., 1986)and cyclin B (Pines and Hunt, 1987), were discovered to periodicallyaccumulate in mitotic cells, and thus a role in the mitotic process wasconsidered possible (Evans et al., 1983) even though the biochemicalbasis was unclear. Results of genetic and biochemical analysis nowsupport a role for certain cyclins in meiosis and mitosis.Microinjection of clam or sea urchin cyclin B1 mRNA into Xenopus oocytes(Pines and Hunt, 1987); Westendorf et al., 1987) is reportedlysufficient to drive the cell through meiosis I and II, and cyclin B maybe the only protein whose synthesis is required for each mitotic cyclein early Xenopus embryos (Murray and Kirschner, 1989). Conversely,destruction of cyclin B1 and B2 mRNA may cause fertilized Xenopus eggsto arrest after DNA replication but before mitosis (Minshull et al.,1989). Besides Xenopus, in the yeasts S. pombe and S. cerevisae cyclin Breportedly plays a role in regulating transit through mitosis (Hagan etal., 1988; Ghiara et al., 1991; Surana et al., 1991; Booher and Beach,1987; Booher et al., 1989; Hagan et al., 1988; Ghiara et al., 1991;Surana et al., 1991) by exerting mitotic control over activation of ap34 CDC2 protein kinase (reviewed in Nurse, 1990; Cross et al., 1989).In the latter case, CDC2 kinase is reportedly not catalytically activeas a monomer, but following binding to the cyclin B and a series ofphosphorylations and dephosphorylation steps, the kinase activity isgenerated (Simanis and Nurse, 1986; Draetta and Beach, 1988; Pondaven etal., 1990; Solomon et al., 1990; Gould and Nurse, 1989; Enoch and Nurse,1990; Solomon et al., 1992).

Cyclin B-dependent activation of a p34 CDC2 kinase may also be necessaryto initiate mitosis in certain somatic cells (Nurse, 1990; Cross, 1989;Maller et al., 1991), but activation alone may not be the only eventrequired (Lamb et al., 1990; Osmani et al., 1991; Amon et al., 1992;Sorger et al., 1992). S. cerevisiae apparently has a CDC2 homologuetermed CDC28. The CDC2 and CDC28 gene products appear to be structurallysimilar (Lorincz & Reed, 1984; Hindley & Phear, 1984) and functionallyhomologous (Beach et al., 1982; Booher & Beach 1987). They encode aserine/threonine protein kinase that is the homolog of the 34 kDaprotein kinase in vertebrate and invertebrate mitosis promoting factor(MPF; Lee & Nurse, 1987; Arion et al., 1988; Dunphy et al., 1988;Gautier et al., 1988; Labbe et al., 1988). CDC28 may require differentcyclins for the cell cycle transitions at G2/M and at G1/S: namely, atG2/M CDC28 reportedly binds to and is activated by B-type cyclins(Ghiara et al., 1991; Surana et al., 1991), while at G1/S CDC28 isreportedly activated by CLN-type cyclins (i.e., CLN1, CLN2 and CLN3;Sudbery et al., 1980; Nash et al., 1988; Cross, 1988, 1990; Hadwiger etal., 1989; Richardson et al., 1989; Wittenberg et al., 1990).

CLN1 and CLN2 cyclins are periodically expressed during the cell cycle,peaking in abundance at the GI/S transition point (Wittenberg et al.,1990; Cross and Tinkelenberg, 1991) and accumulation of the CLN proteinsin yeast cells may be rate limiting for the transition from G1 into Sphase of the cell cycle.

For the purposes of the present disclosure, the term "CDC proteinkinase" is used synonymously with the recently adopted "cell divisionkinase (CDK)" nomenclature.

The p34 CDC2 kinase activity apparently oscillates during the cell cycle(Mendenhall et al., 1987; Draetta & Beach; 1988; Labbe et al., 1989;Moreno et al., 1989; Pines & Hunter, 1990), and this oscillation ofactivity is not attributable to variations in the amount of the CDC2gene product present in cells (Durkacz et al., 1986; Simanis & Nurse,1986; Draetta & Beach, 1988). Rather, CDC2 kinase activity appears to beinfluenced by interactions of the kinase with other proteins, including(as discussed above) the cyclins (Rosenthal et al, 1980; Evans et al.,1983; Swenson et al., 1986; Draetta et al., 1989; Meijer et al., 1989;Minshull et al., 1989; Murray & Kirschner, 1989; Labbe et al., 1989;Soloman et al., 1990; Gautier et al., 1990; reviewed in Murray &Kirschner, 1989; Hunt, 1989). Apparently an association between a p34CDC2 protein and a B-type cyclin is necessary for the activation of thep34 kinase at the onset of mitosis in a wide variety of organismsincluding yeast (Booher & Beach, 1987; Hagan et al., 1988; Moreno etal., 1989; Soloman et al., 1988; Booher et al., 1989; Surana et al.,1991; Ghiara et al., 1991) and humans (Draetta & Beach, 1988; Pines &Hunter, 1989; Riabowol et al., 1989).

In budding yeasts a major control decision point in cell proliferationreportedly occurs during G1, i.e., at a point termed START, where entryof cells into S phase is restricted until certain conditions have beensatisfied (Hartwell, 1974). The START transition appears to require aCDC28 or cdc2 gene product (Hartwell et al., 1973, 1974; Nurse & Bisset,1981), but the biochemical pathways that activate CDC28 at START are notcompletely understood. The latter pathways may involve the CLN1, CLN2and CLN3 cyclins and activation of CDC28 because cells deficient in allthree CLN proteins arrest at START; and although they continue to growthey are unable to enter S phase (Sudbery et al., 1980; Nash et al.,1988; Cross, 1988, 1990; Hadwiger et al., 1989; Richardson et al., 1989;Wittenberg et al., 1990). CLN2, and probably CLN1 and CLN3, may formcomplexes with CDC28 kinase prior to or at START (Wittenberg et al.,1990). The CLN1 and CLN2 oscillates during the cell cycle, but maximallevels are reportedly observed in late G1 (i.e., rather than late G2;Wittenberg et al., 1990).

Little is currently known about the biochemical pathways that controlthe start of DNA synthesis in higher eukaryotic cells or the extent towhich these pathways resemble those in yeast. However, in human cells(as in budding yeast) the predominant mode of control of cellproliferation appears to occur during the G1 phase of the cell cycle(Zetterberg & Larson, 1985; Zetterberg, 1990). The kinetics of passagethrough G1 in mammalian cells suggest a single decision point, termedthe restriction point, that regulates commitment of a cell to initiateDNA synthesis (Pardee, 1974). Prior to the restriction point, progressthrough G1 is sensitive to the growth state of the cell (e.g., reducingthe rate of protein synthesis or removing a growth factor apparently maydelay entry into S phase and can even cause cell cycle arrest), however,after the restriction point the cell cycle becomes substantially lessresponsive to these signals (reviewed in Pardee, 1989). Unlike yeasts,CDC2 cyclin appears to be diversified into a small protein family inmammalian cells (Paris et al, 1991; Elledge and Spotswood, 1991; Tsai etal., 1991; Koff et al., 1991) and CDC2/28 activities may also be splitamong several different kinase family members (Fang and Newport, 1991).Certain cyclins may have roles in G1 regulation in higher eukaryotessimilar to those reported in yeast. For example, cyclin A synthesisreportedly begins late in G1 and it may activate both p34 CDC2 andcertain related p33 CDK2 kinases (Giordano et. al., 1989; Pines andHunter, 1990; Marraccino et al., 1992; Tsai et al., 1991 ). Inhibitionof cyclin A function may also reportedly block a START-like function ofS phase in certain cells (Girard et al., 1991) and cyclin A reportedlyis able to associate with certain transforming and growth suppressingfactors (Hunter and Pines, 1991). However, despite these apparentresults supporting a role for cyclin A in regulating a START-likefunction in higher eukaryotes, there are also some reasons to doubt thatcyclin A is functionally homologous with budding yeast CLN proteins.Several laboratories have recently identified two novel cyclins inmammalian cells that are not present in yeasts, i.e., cyclin C andcyclin D. The cyclin D gene was reported as a gene induced by CSF-1 inmurine macrophages in late G1 (Matshushime et al., 1991) and the genemay have a chromosomal location at a breakpoint subject to possiblerearrangement in human parathyroid tumor (Motokura et al., 1991). CyclinC, as well as cyclin D, have also been reportedly identified in humanand Drosophila cDNA libraries by screening for genes capable ofcomplementing mutations in S. cerevisae CLN genes (Laheu et. al., 1991;Lew et al., 1991; Leopold and O'Farrell, 1991; Xiong et al., 1991).While the results are consistent with G1 functions for cyclin C andcyclin D, cyclin B (a mitotic cyclin) was also found to be capable ofrescuing the latter S. cerevisae CLN mutants, indicating that yeastcomplementation assays may not necessarily identify cyclins that performsimilar functions in higher eukaryotic cells.

The similarities between the restriction point in mammalian cells andSTART in yeast has suggested a possible role for a p34 CDC2 kinase. Insupport of this hypothesis, a human CDC2 gene has been found that may beable to substitute for the activity of an S. pombe cdc2 gene in both itsG1/S and G2/M roles (Lee & Nurse, 1987). Also, cell fusion experimentsoffer circumstantial evidence in support of the hypothesis (Rao &Johnson, 1970) since a diffusible trans-acting factor is reportedlyinvolved in activation of DNA synthesis when S phase cells were fused toG1 cells. However, the relationship between the latter S phase activatorand the p34 CDC2 kinase remains unclear. Recently cyclin-CDC2 complexeshave reportedly been isolated from human S phase cells and shown to beactive in inducing SV40-DNA replication when they were added to extractsof G1 cells (D'Urso et al., 1990). Antisense oligonucleotides directedagainst the human CDC2 mRNA are reportedly inhibitory for humanPHA-activated T cells at entry to S phase (Furakawa et al., 1990). Inother higher eukaryotic cells it has been reported that depletion ofCDC2 protein from Xenopus extracts can block DNA replication (Blow &Nurse, 1990). Despite recent suggestive reports, the pathway thatactivates p34 kinase during the G1 phase of the human cell cycle is notcurrently understood.

By analogy with the CLN-dependent activation of CDC28 at START in yeast,it is possible that specific G1 cyclins may play a role in regulatingthe human p34 kinase during the G1 to S phase transition. To test thisidea experiments were conducted herein to determine whether human cellscontain specific cyclins that can replace the yeast S. cerevisae CLNproteins. This assay identified a new human cyclin, cyclin E.

SUMMARY OF THE INVENTION

The invention provides isolated nucleic acid molecules capable ofhybridizing under stringent conditions to the nucleotide sequenceresiding between positions 1 and 1185 of the human cyclin E cDNAsequence shown in FIG. 2. Such nucleic acid molecules preferably encodecyclin E polypeptides capable of binding and activating a cell divisionkinase (e.g., CDC2, CDC28, CDK2-XL, CDC2-HS, and CDK2-HS). The cyclin Epolypeptide is typically also capable of shortening the G1 phase of thecell cycle. The invention also provides polypeptides encoded by theaforesaid nucleic acid molecules, and immunologic binding partnerscapable of specifically binding the polypeptides.

Cyclin E functions specifically during the late GI and early S phases ofthe cell cycle by binding and activating a CDC2 related protein kinase,CDK2. The levels of the cyclin E/CDK2 polypeptide complexes are cellcycle-regulated, and peak in abundance in late G1 phase of the cellcycle. Constitutive expression of cyclin E in cells is alone sufficientto shorten the G1 phase of the cell cycle and promote cell growth.Increasing or decreasing the levels of cyclin E in a cell increase ordecreases cell growth, respectively. Detecting the levels of cyclin E incells such as tumor cells may provide information on their rate ofgrowth. Rearrangement of the location of cyclin E at chromosomalbreakpoints may change the rate of cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show complementation of the triple cl n deletion byhuman cyclin E: S. cerevisae strain 589-5 contains deletions of thechromosomal CLN1, -2, and -3 genes and contains the GAL1-CLN3 gene on amulticopy episome. It was transformed with the pADNS expression vectoror the pADNS vector containing a human cyclin E cDNA. Transformants, andthe parental strain, were streaked on galactose and glucose and grownfor 3 days at 30° C.

FIGS. 2A-2C (SEQ. ID. NOS. 1-2) show the sequence of cyclin E: DNA andpredicted protein sequence of the cyclin E cDNA that complemented thetriple cln deletion.

FIGS. 3A and 3B show alignment of the protein sequences of human cyclinsA, B, and E: Protein sequences of cyclins A, B, and E were aligned tomaximize homology. Bold letters indicate identical amino acids. Aminoacid shared by all three cyclins are shown in bold type above the threesequences. The area highlighted by double bold lines is a domain highlyconserved among all known cyclins (the "cyclin box"). The domainhighlighted by single bold lines is the mitotic destruction motif sharedby all A- and B-type cyclins.

FIG. 4 shows efficiency of rescue of the triple cln deficiency by humancyclins E and B in CDC28 or cdc28-13 strains: All strains tested werecln1⁻ cln2⁻ cln3 (pGAL-CLN3); these strains were transformed with theindicated vector plasmids, pADNS or pADANS, or the vector plasmidscontaining either human cyclin E or B by selecting for leucineprototrophy. The vector pADNS uses the yeast ADH promoter for expressionof the cDNA. The vector pADANS is identical to pADNS except that theexpressed protein is fused at its amino terminus to the first 10 aminoacids of the ADH protein. No transformants could be obtained with theplasmid pADNS-CYC B, suggesting it was lethal. The number of viablecolonies in an inoculum of stationary phase culture in galactose wasdetermined by serial dilution followed by 4-5 days growth on bothgalactose- and glucose-containing medium at 30° C. The platingefficiency is defined as the number of glucose-viable colonies dividedby the number of galactose-viable colonies. Only colonies resulting fromplasmid bearing cells are used in the calculation. Both YC and YEP mediawere used with comparable results. The vector and pDANS-CYC E valueswere determined in four experiments using two different pairs of CDC⁺and cdc28-13 strains (one CDC⁺ and one cdc28-13 strain tested inparallel in each experiment). The pADANS-CYC E values come from a singleexperiment. The cyclin B values were determined in two experiments, bothwith the same pair of CDC⁺ and cdc28-13 strains. All strains wereisogenic. The ranges of values for the CDC⁺ strains were: for pADNS-CYCE, 0.12-0.4; for pADANS-CYC B, 0.19-0.33; for the cdc28-13 strainpADNS-CYC E, 0.0006-0.008; and for pADANS-CYC B, 0.2-0.4. With bothcyclin E plasmids the colony sizes for cdc28-13 strains on glucosemedium were significantly smaller than colony sizes for CDC⁺ strains;for the cyclin B plasmid the colony sizes were similar in the CDC⁺ andcdc28-13 strains.

FIG. 5 shows a yeast strain in which CDC28 is defective for START butnot G2/M: Yeast strain 1238-14C-cycE has the following relevantgenotype: cln1⁻ cln2⁻ cln3⁻ cdc28¹³ (pADH-cycE-TRP1,pGAL-CLN3-URA3).Shown are cyclin-cdc28-13 complexes that form on either galactose orglucose and the functional activity of those complexes at either 30° or38° C. CLB is the nomenclature used to designate the S. cerevisaehomologs of the B-type cyclins. On glucose at 30° C. this strain isdefective for START but not G2/M.

FIG. 6 shows efficiency of rescue of the triple cln deficiency in acdc28-13 strain by human cyclin E in conjunction with human CDC2 orhuman CDK2: A strain of genotype cln1⁻ cln2⁻ cln3⁻ cdc28-13(pGAL-CLN1/URA3) was cotransformed with either pMAC-TRP1-CYC E andpADNS-LEU2-CDC2-HS or with pMAC- TRP1-CYC E and pADNS-LEU2-CDK2-HS.Transformants were selected for leucine, tryptophan, and uracilprototrophy on galactose. Two independent transformants were grownnonselectively overnight, and plasmid loss events were identifiedfollowing colony purification. This resulted in the generation ofisogenic sets of strains, either containing both cyclin E and CDC2-HS(or CDK2-HS) or either gene alone. Two such sets were generated for eachcotransformation. The twelve strains were tested in the quantitativeplating assay described above (see legend to FIG. 4) except that platingefficiencies were measured both at 30° and 38° C. Strains containing thesame plasmid combinations behaved very similarly, and their data arepooled in the table. Note that unlike cyclin E plasmids used in theexperiments in FIG. 4, the pMAC-TRP1-CYC E plasmid used in theseexperiments gives essentially no rescue of the c1n1-c1n2-c1n3-cdc29-13strain.

FIGS. 7A-7C show cyclin E can bind and activate the p34 cdc2 kinase inextracts from human G1 cells: Extracts from newborn MANCA human G1 cellswere mixed with GT-cyclin E-Sepharose beads, GT-Sepharose,p13-Sepharose, or blank Sepharose beads. In FIG. 7A, the bound proteinswere immunoblotted with anti-peptide antiserum against the carboxyterminus of human CDC2. In lanes labeled "+" the Sepharose beads hadbeen incubated with the human G1 extract. In lanes labeled "-" mockincubations with buffer were performed. The arrow indicates the bound 34kDa protein that reacts with the C-terminal antibodies. In FIG. 7B, thebeads were assayed for histone H1 kinase activity. Arrows indicate themobility of histone H1 and GT-cyclin E fusion protein markers. In thelane labeled "cycE-IP" the proteins associated with the GT-cyclinE-Sepharose beads were released with free glutathione andimmunoprecipitated with a cyclin E antiserum. In FIG. 7C, the proteinsreleased from either the GT-Sepharose or GT-cyclin E-Sepharose by freeglutathione were immunoprecipitated with an affinity-purifiedC-terminus-specific p34 CDC2 anti-peptide antiserum. Theimmunoprecipitates were tested for H1 kinase activity.

FIGS. 8A-8B show immunoprecipitation of an H1 kinase activity from HeLacells using anti-cyclin E antibodies: In FIG. 8A, an antiserum raised inrabbits against the GT-cyclin E fusion protein was used toimmunoprecipitate in vitro translated human cyclins E, A, and B. Lanes1-3: in vitro translation products of human cyclins E, A, and Brespectively. Lanes 4-6: the immunoprecipitates using the cyclin Eantiserum of human cyclins E, A and B respectively. In FIG. 8B, extractsfrom exponentially growing HeLa cells were immunoprecipitated withnormal rabbit serum (NRS), anti-cyclin E serum (CYCE), and anti-cyclin Aserum (CYCE). The immunoprecipitates were tested for H1 kinase activity.Note the autophosphorylation of a 45 kDa protein within the cyclin Eimmunoprecipitates. This protein comigrates with cyclin E proteinproduced by in vitro transcription/translation of the cyclin E cDNA.

FIGS. 9A-9E show differential levels of cyclin E-kinase complexes indifferent subpopulations of exponentially growing MANCA cells that werefractionated by centrifugal elutriation into different stages of thecell cycle, as described in Example 8.

FIG. 9A shows graphically the DNA content (ordinate) of exponentiallygrowing MANCA cells measured cytofluorimetrically in differentelutriated fractions (abscissa).

FIG. 9B shows graphically the level of cyclin E H1 histone kinaseactivity in the elutriated fractions of FIG. 9A.

FIG. 9C shows graphically the level of cyclin A H1 histone kinaseactivity in the elutriated fractions of cells of FIG. 9A.

FIG. 9D shows graphically the DNA content (ordinate) of MANCA cells indifferent elutriated cell fractions released into the G1 phase of thecell cycle for 3, 4, 5, 6, or 7 hours after nocodazole-inducedmetaphase.

FIG. 9E shows a bar graph depicting the levels of cyclin A and cyclin Eassociated H1 histone kinase activity.

FIGS. 10A-10B discussed in Example 8, show the levels of cyclin E, asdetermined by measuring H1 kinase activity, in immunoprecipitates ofquiescent, growing, or differentiating, rat 208F and PC-12 cells.

FIG. 10A shows an autoradiogram of ³² P-labeled H1 histone. Thephosphorylation of H1 histone was catalyzed by immunoprecipitates of208F cells grown in 10% or 0.1% calf serum. The level of cyclinE-associated kinase activity was markedly reduced in quiescent (0.1% CS)cells as compared to growing cells (10% CS).

FIG. 10B shows an autoradiogram of ³² P-labeled H1 histone. Thephosphorylation of H1 histone catalyzed by immunoprecipitates wasdetermined for PC-12 cells grown in the presence or absence of nervegrowth factor. The level of cyclin E-associated kinase activity wasmarkedly reduced in differentiated (quiescent) cells (-NGF) as comparedto rapidly proliferating cells (+NGF).

FIGS. 11A-11E discussed in Example 9, show the results of studiesdesigned to investigate increased levels of constitutively expressedcyclin E in Rat-1 cells transduced with either a retroviral vectorencoding cyclin E (LXSN-cyclin E), or the LXSN vector as a negativecontrol.

FIG. 11A shows an autoradiograph of a Western immunoblot of Rat-1cellular lysates transduced with either LXSN-cyclin E (lane 2) or, as anegative control, the LXSN vector alone (lane 1). Increased levels ofcyclin E, as measured by histone H1 kinase activity, were visible inLXSN-Cyclin E transduced cells relative to the control.

FIG. 11B shows an autoradiogram of ³² P-labeled histone H1 catalyzed bycyclin E-associated kinase in cellular immunoprecipitates of LXSN-cyclinE transduced Rat-1 cells (lane 2) or LXSN transduced control Rat-1 cells(lane 1). Increased cyclin E expression was visible in cellularimmunoprecipitates of LXSN-cyclin E transduced Rat-1.

FIG. 11C graphically presents the results of flow cytometric measurementof nuclear DNA content in Rat-1 cells transduced with either LXSN(Rat-1/control) (FIG. 11C) or the LXSN-cyclin E retroviral vector(Rat-1/cyclin E) (FIG. 11D), as well as the calculated fraction of thecells in each cell subpopulation that was in the G1, S, or G2/M phasesof the cell cycle.

FIG. 11D graphically presents the results of immunochemical detection ofBrdU (5-bromodeoxyuridine) incorporation into nuclear DNA of Rat-1 cellstransduced with LXSN-cyclin E (solid diamonds) or LXSN (open squares) asa function of time after removing a mitotic block. Only cellssynthesizing DNA (S-phase cells) incorporate BrdU and score positive inthis assay, and so the assay measures the rate at which cells transitionfrom the conclusion of one mitosis into DNA synthesis for the next roundof mitosis. The results show that LXSN-cyclin E transduced cellstransition from mitosis into S-phase more rapidly than LXSN-transducercontrol cells.

FIG. 12, discussed in Example 10, shows the results of a study analyzingproteins that are associated with cyclin E in exponentially growingMANCA cells at different stages in the cell cycle. The cyclinE-associated proteins were purified by immunoprecipitation withanti-cyclin E antibodies and SDS-PAGE.

FIG. 13, discussed in Example 10, shows autoradiograms of Westernimmunoblots prepared following SDS-PAGE of immunoprecipitates preparedfrom MANCA cell extracts that were immunoprecipitated with anti-CDC2("αCDC2"), anti-CDK2 ("αCDK2"), or control serum ("-"). The results showthe specificity of the anti-CDC2 and anti-CDK2 antibodies used in FIGS.9-12 above. The immunoblots were visualized by reacting the gels withanti-CDC2 or anti-CDK2 followed by ¹²⁵ I-Protein A. Theimmunoprecipitates in lanes 1 and 2 were prepared using whole cellextracts; those in lane 3 and 4 were control extracts that wereprecleared of CDC2; those in lanes 5 and 6 were precleared of CDK2; andlane 7 was an extract of MANCA cells arrested at the G1/S boundary. Thepositions migrated by protein molecular weight standards is asindicated.

FIGS. 14A-14B, described in Example 10, show Western immunoblotsdetecting complexes of CDC2 and CDK2 with cyclin E in exponentiallygrowing MANCA cells and cells arrested at the G1/S boundary withaphidicolin by methods described in Example 10.

FIG. 14A shows an immunoblot of purified cyclin E:CDC2 complexesseparated into its two constituent proteins on SDS-PAGE and visualizedby immunoblotting with antibodies specific for the C-terminus of humanp34 CDC2. Lanes 1 and 8 were negative control samples prepared from anincubation of cell extracts with αPI; lanes 2 and 7 were negativecontrols from an incubation of cell extracts with Sepharose beads(SEPH); lanes 3 and 6 show cyclin E:CDC2 complexes purified by affinitychromatography or anti-p34 CDC2 Sepharose; lanes 4 and 5 show cyclinE:CDC2 complexes purified by affinity chromatography on anti-cyclinE-Sepharose; lanes 9-11 labeled "-" are negative control samplesprepared in a manner identical to lanes 1-8, but without cell extract.

FIG. 14B shows an immunoblot of purified cyclin E:CDK2 complex separatedinto its two constituent proteins and CDK2 visualized by immunoblottingwith antibodies specific for the C-terminus of human CDK2. Theabbreviations used are as indicated in FIG. 14A. The location onSDS-PAGE of CDK2 in nonaffinity-purified SDS-PAGE purified cell extract("EX") from cells at the G1/S boundary is shown in lane 8.

FIGS. 15A-15C, described in Examples 11-12, shows the results of studiesdesigned to determine the level of expression of cyclin E and abundanceof the cyclin E:CDK2 complex at different stages in the cell cycle.

FIG. 15A shows graphically the DNA content (ordinate) in variousfractions of elutriated MANCA cells as determined by flow cytometry ofpropidium iodide stained nuclei.

FIG. 15B shows graphically the ¹²⁵ I-Protein A CPM bound by the CDK2polypeptide bands immunoblotted in FIG. 15C.

FIG. 15C shows Western immunoblots measuring the level of expression ofcyclin E in each elutriated subpopulation of cells (from FIG. 15A) asdetermined by immunoaffinity purification of cyclin E:CDK2 complexeswith anti-cyclin E-Sepharose followed by SDS-PAGE, and visualization ofthe proteins in the complex by Western immunoblot analysis usinganti-CDK2, ¹²⁵ I-Protein A, and autoradiography.

FIG. 15D shows graphically the ¹²⁵ I-Protein A CPM bound by the cyclin E("cycE") polypeptide bands immunoblotted in FIG. 15E.

FIG. 15E shows Western immunoblots measuring the level of expression ofcyclin E in elutriated subpopulations of cells from FIG. 15A by themethods described in FIG. 15C, but using anti-cyclin E and ¹²⁵ I-ProteinA to visualize the level of cyclin E instead of anti-CDK2.

FIG. 15F shows graphically the level of ¹²⁵ I-Protein A bound byanti-cyclin E:cyclin E bands in Western immunoblots as a function of theamount of cellular extract (elutriated fraction 3 extract) used in themethod of FIGS. 15B-15E. The results show that increasing the amount ofcyclin E increased the amount of signal in the immunoassay for cyclin E.

FIGS. 16A-16C, described in Example 13, show the results of studiesdesigned to investigate the molecular association of cyclin E with CDC2and CDK2; assembly of cyclin E:CDC2 or cyclin E:CDK2 complexes in vitro;and activation of phosphorylase kinase activity following association ofthe kinases with cyclin E.

FIG. 16A shows the results of experiments assaying phosphorylase kinaseactivity in cyclin E:CDK2 complexes immunoprecipitated with antibodyspecific for CDK2 (Anti-CDK2). The complexes were formed in the cellextracts of hydroxyurea-arrested cells (HU) and in extracts of G1 phasecells (G1 extract) in the absence (0) or presence of differing amounts(5,1,0.2) of recombinant cyclin E. Kinase activity in theimmunoprecipitates was determined using histone H1 as a substrate,SDS-PAGE, and phosphor imaging of the ³² P-labeled histone H1 bands inthe gels. The results show that the addition of cyclin E to G1 cellextracts activated latent kinase activity in the extracts in adose-dependent manner, i.e., the level of kinase activity measured wasdependent upon the amount of cyclin E added.

FIG. 16B shows the results of experiments assaying phosphorylase kinaseactivity in cyclin E complexes. The experiments were conducted asdescribed in FIG. 16A, above, but using antibodies specific for cyclin E(Anti-cyclin E) instead of Anti-CDK2. The results confirm thosepresented in FIG. 16A, above: namely, cyclin E activates a latent CDCkinase activity in extracts of G1 cells in a dose-dependent manner.

FIG. 16C shows the results of experiments designed to assay for thephosphorylase kinase activity in cyclin E:CDC2 complexes. Theexperiments were conducted as described in FIG. 16A, above, but usingantibodies specific for CDC2 (Anti-CDC2). The results show minimal to noCDC2 kinase activity in G1 extracts of cells even when cyclin E wasadded.

FIG. 17 graphically depicts the results obtained in FIGS. 16A, 16B, and16C, above. The results obtained with each respective G1 cell extractimmunoprecipitate (CDC2, solid/left-most bars in set of three bars;CDK2, hatched bars/middle of each set; or, cyclin E, shaded/right-mostbars in each set of three) is expressed as a percentage of the phosphorimaging signal obtained with the kinase in the HU-cell extractimmunoprecipitate (i.e., 100%; % hydroxyurea H1 kinase). The numbers atthe top of each bar are the maximal value recorded in percent (%). Theresults show that cyclin E activated a latent CDK2 kinase activity inthe G1 cell extracts and the activity of the kinase was dependent uponthe amount of cyclin E added (i.e., expressed as the fold-dilution ofthe cyclin E added to the HU extract; "fold cyclin E in HU extract").The results indicate that the availability of cyclin E is a factorcontrolling phosphorylase kinase activity during the G1 phase of thecell cycle.

FIGS. 18A-18B, described in Example 14, graphically represent theeffects of serum growth factors on the rate at which LXSN-cyclinE-transduced Rat-1 cells and LXSN-transduced control cells initiate DNAsynthesis following nocodazole-arrested mitosis. The incorporation ofBrdU into nuclear DNA was measured in LXSN-cyclin E-transduced(RAT1/cyclin E) or LXSN-transduced (RAT1/LX) control cells as a functionof time after the nocodazole mitotic block. Only cells synthesizing DNA(i.e., S-phase cells) incorporate BrdU into DNA and scoring the numberof nuclei in a cell culture (i.e., % labelled nuclei) can thus be usedto evaluate the rate of transition of the cells from G1 into theS-phase.

FIG. 18A shows the results of experiments designed to evaluate the rateat which LXSN-cyclin E-transduced (RAT 1/cyclin E) or LXSN-transduced(RAT 1/LX) control cells initiate DNA synthesis following release of anocodazole block. The results show that the cyclin E-transduced cellsinitiated DNA synthesis more than 2-3 hours earlier thancontrol-transduced cells, and the rate of nuclear labeling (i.e.,initiation of DNA synthesis) was also greater in the cyclin E-transducedcells (as evidenced by the differing slopes of the two curves).

FIG. 18B shows the results of experiments designed to evaluate thegrowth factor dependence for initiating DNA synthesis in LXSN-cyclinE-transduced and LXSN-transduced control cells. Both types of cells werecultured in medium containing either 1% or 0.1% bovine calf serum. Theresults show that a) LXSN-cyclin E-transduced cells initiated DNAsynthesis more rapidly initiation of DNA following release of thenocodazole block in either 1% or 0.1% serum, and b) cyclin E-transducedcells were less dependent on growth factors than LXSN-transduced controlcells as evidenced by their more rapid initiation of DNA synthesis inlow serum (i.e., the LXSN-cyclin E-transduced cells initiated DNAsynthesis more than 6-8 hours earlier than LXSN-transduced cells in 0.1%serum), and the cyclin E-transduced cells also proliferated more rapidlyin the low serum conditions than the control (i.e., as determined bycomparing the slopes of the the two transduced cell types grown in 0.1%serum).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A new human cyclin, named cyclin E, was isolated by complementation of atriple cln deletion in S. cerevisae. Cyclin E showed geneticinteractions with the CDC28 gene suggesting that it functioned at STARTby interacting with the CDC28 protein. Two human genes were identifiedthat could interact with cyclin E to perform START in yeast containing acdc28 mutation. One was cdc2-HS and the second was the human homolog ofXenopus CDK2. Cyclin E produced in E. coli, bound and activated the CDC2protein in extracts from human G1 cells, and antibodies against cyclin Eimmunoprecipitated a histone H1 kinase from HeLa cells. The interactionsbetween cyclin E and CDC2, or CDK2, may be important at the G1 to Stransition in human cells.

The invention provides nucleic acid molecules capable of hybridizingunder stringent conditions to the human cyclin E cDNA shown in FIG. 2from position 1 to 1185. Although only a single (+) strand of the cDNAis shown in FIG. 2, those skilled in the art will recognize that itscomplementary (-) strand is thereby disclosed as well. By nucleic acidmolecule is meant DNA, RNA, and/or synthetic nucleotide sequences suchas oligonucleotides that are the same as, homologous with, orcomplementary to, at least one helical turn (about 10 to 15 nucleotides)of the illustrated cyclin E nucleotide sequence. The invention providesmore than three cyclin E cDNAs resulting from alternative splicing ofcyclin E mRNAs, genetic polymorphism, and translocation intumorigenesis. Those skilled in the art will recognize that the membersof this closely related group of cyclin E nucleic acids are readilyidentified by their ability to hybridize under stringent conditions withall or portions of the nucleotide sequence of FIG. 2 or itscomplementary (-) strand. By capable of hybridizing under stringentconditions is meant annealing of a nucleic acid molecule to at least aregion of the disclosed cyclin E nucleic acid sequence (whether as cDNA,mRNA or genomic DNA) or to its complementary strand under standardconditions, e.g., high temperature and/or low salt content, which tendto disfavor hybridization of noncomplementary nucleotide sequences. Asuitable protocol (involving 0.1×SSC, 68° C. for 2 hours) is describedin Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, ColdSprings Harbor Laboratory, 1982, at pages 387-389. Such hybridizingnucleic acid molecules may be related to the disclosed sequence bydeletion, point mutation, base substitution, frameshift, alternativeORFs, mRNA splicing and processing, or post-transcriptional modification(e.g., methylation and the like). For example, antisense nucleic acidsare provided having nucleotide sequences complementary to the cyclin Esequence and characterized by the ability to inhibit expression of acyclin E gene, e.g., by binding and inhibiting translation of a cyclin EmRNA. Antisense nucleic acids may be encoded within a host cell, e.g.,following transduction or transfection of the cell with a vector DNA orRNA sequence encoding an antisense nucleic acid, or, alternatively, theantisense nucleic acids may be synthetic oligonucleotides. Suchantisense oligonucleotides are introduced into cells by a variety ofmeans, e.g., with retroviral vectors encoding antisense mRNA in thecell, or by fusing the cell with liposomes containing an antisenseoligonucleotide and the like. The subject antisense nucleic acidmolecules are characterized by their ability to hybridize understringent conditions with the illustrated cyclin E nucleic acid, itscomplementary strand, 5' transcription regulatory regions of a cyclin Egene, or translation regulatory regions of a cyclin E mRNA.

The isolated nucleic acids of the invention preferably encode cyclin Epolypeptides. Such polypeptides are not necessarily encoded by theaforesaid isolated nucleic acid molecules, since those skilled in theart will recognize that the disclosed cyclin E nucleotide sequencepermits construction of a variety of synthetic polypeptides. Suchsynthetic polypeptides may vary in length (e.g., from about 5 aminoacids to many hundreds of amino acids) and be constructed correspondingto selected regions of the encoded cyclin E polypeptide. The subjectcyclin E polypeptides thus encompass isolated cyclin E polypeptides(i.e., found in normal cells), mutant polypeptides (e.g., resulting frommutagenesis, or found in tumor cells), and chemically modifiedpolypeptides (e.g., having one or more chemically altered amino acids,in which case a designated amino acid can be convened into another aminoacid, or chemically substituted or derivatized and the like). Functionalsites in the cyclin E polypeptides are identified by constructingmutants of the cyclin E nucleic acid, e.g., and testing the constructsfor expression products having altered functional properties such asfailure to bind or activate a CDC protein kinase, or failure to advancethe cell cycle. Particularly useful for constructing such mutants areregions of conserved nucleotide or amino acid sequence, e.g., conservedbetween cyclins A, B, C, D, and E, or conserved among the members of thecyclin E family. Conserved regions of cyclin E are functional andprotein-structural regions of the polypeptide.

In an illustrative preferred aspect, expression of a cyclin Epolypeptide in a cell allows levels of cyclin E in the cell to rise to apoint where cyclin E binds and activates a CDC protein kinase, andeliminates certain growth factor and serum requirements for progressionof the cell through the G1 phase of the cell cycle. As a result, the G1phase of the cell cycle is shortened. The G1 phase commonly lasts about8 to about 12 hours, and expression of a cyclin E polypeptide in a cell(or exposure of a cell to a cyclin E polypeptide) may shorter G1 phaseby about 1 hour to many hours. That a cyclin of the invention shortensthe G1 phase of the cell cycle can be readily determined by thoseskilled in the art by using a model test system such as that providedbelow in the Examples, e.g., by the "598-5" or "1238-14C" strains ofyeast. Alternatively, a mammalian cell such as an NIH3T3 cell may betransfected or transduced with an expression vector containing a cyclinE-hybridizing nucleic acid and the length of the G1 phase of the 3T3cell cycle can then be determined in kinetic cell cycle assays such asthose illustrative examples provided below. For instance, those skilledin the art will recognize that progression of cells from M to S phasecan be measured (i.e., in hours and minutes) by determining tritiatedthymidine or bromodeoxyuridine (BrdU) incorporation. Cyclin E nucleicacid, when transfected or transduced into test cells, induces either afaster progression of the cells from the M phase to the S phase; or aprogression of the cells from M to S without requiring exogenousstimulae, i.e., serum or growth factors and the like. In either case,introducing the subject cyclin into the test cell results in ashortening of the G1 phase and a more rapid progression from M to S.

Representative examples of CDC protein kinases to which cyclin E bindsinclude CDC2, CDC28, CDCK2-XL, CDC2-HS, and CDK2-HS. (Note that in thisterminology "HS" designates Homo sapiens and "XL", Xenopus laevis.) Theinvention also provides methods for identifying and cloning other CDCkinases that are bound and activated by cyclin E (see Example 10,below). Those skilled in the art will understand that synthetic cyclin Epolypeptides may be readily constructed by modifying the disclosed aminoacid sequence and testing for altered functional properties, i.e.,altered binding, activation of a CDC protein kinase, and/or alteredability to shorten the G1 phase of the cell cycle. Such synthetic cyclinE polypeptides are useful competitive and noncompetitive inhibitors of anormal or mutant cyclin E (i.e., derived from a normal or mutant cell)or of its CDC protein kinase binding partner. Such syntheticpolypeptides also include polypeptide antagonists or agonists useful forchanging the functional properties of the cyclin E:CDC protein kinasecomplex, e.g., by increasing, decreasing, or otherwise modifying ormodulating: a) the phosphorylase activity activated by the CDC proteinkinase; b) the activity of the cyclin E, e.g., for activating the CDCprotein kinase; c) the cell cycle promoting activity of the cyclinE:cell division kinase complex; and/or, d) transcriptional regulatoryfactors that bind the 5' region of the cyclin E gene.

Skilled artisans will further understand that the disclosure herein ofrecombinant cyclin E nucleic acids, cells, and in vitro assays provideopportunities to screen for compounds that modulate, or completelyalter, the functional activity of a cyclin E protein or cyclin E nucleicacid in a cell. In this context "modulate" is intended to mean that thesubject compound increases or decreases one or more functional activityof a cyclin E protein or nucleic acid, while "alter" is intended to meanthat the subject compound completely changes the cyclin E protein ornucleic acid functional activity to a different functional activity. Inthis context, an example of a compound that "modulates" the activity ofa cyclin E protein is an inhibitor capable of decreasing the level ofCDC kinase activity following binding of a cyclin E to the CDC kinase;and, an example of a compound that "alters" the activity of a cyclin Eprotein is an agent that induces cyclin E to bind to CDC2 instead of toCDK2.

The screening assays illustrated in the Examples (below) includebiochemical assays (e.g., measuring effects of cyclin E protein on CDC2and CDK2 phosphorylase activity), and cellular in vitro assays (e.g.,measuring the effects of cyclin E expression on cell proliferation). Theillustrative biochemical assays may be particularly useful in screeningfor compounds modulating a cyclin E molecular activity, while thecellular assays may be particularly useful in screening for compoundsaltering a cyclin E activity in a cell. For example, in proliferatingcells cyclin E participates with other cyclins, CDC kinases, growthfactor second messengers, transcription regulatory factors and the likein controlling the proliferative response of a cell to its environment.Those skilled in the art will understand that binding of a ligand at amolecular binding site can be modulated in a direct manner (e.g., byblocking the site), as well as altered in an indirect manner (e.g., byconformational changes induced following binding of a second (different)ligand at a distant site). In this regard, it is likely that the bindingsite specificity of cyclin E for a particular CDC kinase (or some othercellular control factor, as discussed below), can be completely altered(i.e., to bind a different ligand) by agents that bind at distant sitesin the cyclin E polypeptide. Examples of compounds that may be screenedin the latter several assays include at least nucleic acids (e.g., DNAoligonucleotide aptamers that bind proteins and alter their functions),proteins, carbohydrates, lectins, organic chemicals, and the like. Suchscreening assays may be useful for identifying candidate therapeuticagents that may provide drugs useful in animals and humans.

It is still further understood that, due to the significance of cyclin Eand the cyclin E:CDC protein kinase in the cell cycle, innate regulatorymechanisms exist in cells for regulating their activity by binding tocyclin E or to complexes containing cyclin E. Such regulatory factorscan include, at least: a) cofactors that bind to the complex and exertregulatory action by destabilizing or stabilizing the complex; b) agentsthat modulate or alter the activity of the complex by inducingconformational changes in the CDC protein kinase and/or cyclin Epolypeptides as they are bound together in the complex; c)enzymes thatinactivate one or both members of the complex; and, d) cellular controlfactors (e.g., signal transduction second messengers, transcriptionregulatory factors, and the like) that bind cyclin E or cyclin Ecomplexes and modulate or alter functional activity. Thus, artificialpolypeptides can be constructed that control the activity of the cyclinE:CDC protein kinase kinase complexes in the cell by inhibiting orpromoting the activities of such regulatory factors. Those skilled inthe art will recognize that the functional regions of cyclin E representparticularly attractive targets for three-dimensional molecular modelingand for the construction of mimetic compounds, e.g., organic chemicalsconstructed to mimic the three-dimensional interactions between thecyclin E and its CDC protein kinase binding partner. In a particularlypreferred embodiment, the invention provides isolated nucleic acidmolecules that encode artificial cyclin E polypeptides that bind to, butdo not activate, CDC protein kinases.

In other preferred embodiments of the invention, polypeptides areprovided that are encoded by nucleic acids corresponding to thefollowing regions of the cyclin E nucleotide sequence (i.e., regionsthat are conserved between cyclins A, B, and E): namely, a) acarboxy-terminal leucine repeat sequence (i.e., residing betweenpositions 640 and 1185, and more particularly between positions 631 and936, of the cyclin E cDNA shown in FIG. 2A); b) an MRAIL sequence (i.e.,residing between positions 385 and 645 of the cyclin E cDNA shown inFIG. 2A); and c)a C-terminal sequence region (i.e., residing betweenpositions 1048 and 1080 of the cyclin E cDNA shown in FIG. 2A). TheMRAIL sequence is necessary, but not sufficient, for binding to a celldivision kinase.

It is further understood that mutant cyclin E nucleotide sequences maybe constructed from the sequence shown in FIG. 2A. The subject mutantcyclin E nucleotide sequences are recognized by their ability to encodemutant cyclin E polypeptides that may have binding affinity for a CDCkinase (e.g., CDK2) polypeptide that is higher or lower than thatexhibited by a cyclin E polypeptide for a CDC kinase in anon-transformed mammalian cell. The subject mutant polypeptides also mayalter (i.e., increase or decrease) the enzyme activity of the CDK2kinase when the subject mutant cyclin E polypeptide and CDK2 areresident together in a complex. Illustrative examples of changed enzymeactivity include: a )increased or decreased enzymatic activity (e.g.,Kin, Vmax, kcat, and the like); b) changed stability of the kinase inthe complex (e.g., to time-dependent decay of the complex or the enzymeactivity); c) changed susceptibility of the kinase to proteolyticinactivation; d) changed susceptibility of CDK2 to dissociate from thecyclinE:CDK2 complex in response to binding of regulatory factors(discussed above) by the complex; or e) changed sensitivity of thekinase in the complex to competitive or noncompetitive inhibitors.Skilled artisans will recognize a variety of methods by which thesequence in FIG. 2 may be mutated (e.g., with chemical agents orradiation), and by which clones of cells containing the mutated cyclin Enucleotide sequences may be identified and/or selected. The subjectmutant cyclin E nucleotide sequences are useful for modulating oraltering the activity of a cyclinE:CDK2 complex in a cell, e.g., in atumor cell to decrease CDK2 kinase activity and slow cell proliferation,or in a terminally differentiated cell to increase CDK2 kinase andstimulate growth. The subject mutant cyclin E nucleotide sequences maybe introduced using vectors such as the illustrative retroviral vectorsin Example 9.

Artificial cyclin E polypeptides, organic chemical mimetics, antisenseRNA and oligonucleotides, and the like find broad utility as selectiveinhibitors of cell proliferation triggered by growth factors, mitogens,cytokines, and like agents, without inhibiting ongoing reparativemitotic activity in a tissue. Thus, it will be appreciated that thesynthetic polypeptides, mimetics, and antisense embodiments of theinvention will preferably exhibit differential inhibitory activities;e.g., when the subject inhibitor is introduced into two cells, onetriggered by a cytokine to proliferate, and a second undergoing mitosis,the first cell is inhibited but the second cell is not. The subjectsynthetic cyclin E polypeptides of the invention only inhibit cells thatare transitioning from G1 to S, and not those cells that are alreadyactively undergoing mitosis. Thus, those skilled in the art willrecognize that representative examples of utility include inhibitinginduction of immune responses; interrupting clonal expansion (i.e.,either T or B lymphocyte) of an ongoing immune response; inhibitinggrowth factor-induced proliferation of tumor cells or metastatic cellsthat are transitioning from G1 to S; and inhibiting growthfactor-induced tissue hypertrophy (e.g., vascular smooth muscle cellproliferation such as in atherosclerotic plaques, mesenchymalhypertrophy of fibroblasts and connective tissue cells such as inrheumatic joints, and the like). The subject selective inhibitors areconveniently recognized, for instance, by their ability: a) to eitherdecrease (or increase) the levels of cyclin E polypeptide or mRNA in atest cell in vitro (i.e., compared to a control cell of the same type);b) to decrease (or increase) the level of cyclin E that can form acomplex with CDK2; or c) to decrease (or increase) the binding affinityof cyclin E polypeptides for members of the CDK2 family of cell cycledependent kinases in mammalian cells. Skilled artisans will recognizethat measuring a decreased (or increased) activity of a subjectselective inhibitor may be accomplished using asynchronized orsynchronized cell cultures; e.g., in synchronized cultures of cellscyclin E levels and activities are examined during the G1 phase of thecell cycle.

Aspects of the invention include recombinant expression vectors such asviral vectors for mammalian cells (e.g., retroviruses similar to thatillustrated in Example 9, vaccinia virus, adenoviruses, CMV, and thelike), and plasmid or cosmid vectors useful for transfecting andtransducing nucleic acid into prokaryotic and eukaryotic cells.Recombinant expression vectors of the invention are constructed forexample by operably linking a cyclin E nucleic acid to suitable controlsequences. Operably linking is used herein to mean ligating a cyclin Enucleic acid to an expression vector nucleic acid in a manner suitablefor transcription and translation of cyclin E, preferably under apredetermined positive (or negative) regulatory control exerted bycontrol sequences in the expression vector (i.e., containing regulatorysequences capable of driving expression, over-expression, andconstitutive-expression of the cyclin E gene, e.g., promoter, enhancer,operator sequences, and the like). Selectable markers will generally beincluded in the expression vector. Representative examples of suchselectable markers include enzymes, antigens, drug resistance markers,or markers satisfying the growth requirements of the cell. It will alsobe appreciated that in certain cells transfection or transduction withcyclin E nucleic acid will provide a selective proliferative/growthadvantage that will serve as a type of selectable marker (e.g., inmutants blocked for progression of the cell cycle, such as therepresentative yeast strains "598-5" or "1238-14C" described below inthe Examples). The subject expression vectors are useful fortransfecting and transducing cells to produce cyclin E polypeptides,mutant cyclin E polypeptides, and antisense nucleic acids. For instance,aspects of the invention include methods for using the transfected andtransduced cells to produce polypeptides that are able to activate a CDCprotein kinase and advance the cell cycle at the restriction point fromthe G1 phase to the S phase. Several methods are available fordetermining that a polypeptide encoded by a cyclin E nucleic acid iscapable of advancing the cell cycle. Representative examples involvingyeast cells and mammalian cells are described in the Examples, below.

The invention also provides a cell type that has been transduced ortransfected with a cyclin (e.g., cyclin E) expression vector. In onepreferred embodiment a cell constitutively over-expressing cyclin Eproliferates at a rate faster than its parent cell. Cyclin E-transducedhuman diploid fibroblasts illustrated in Example 14, below, were 20-50%smaller in length and width than control-transduced or normal cells.Skilled artisans will understand the advantages in gene therapy of smallrapidly growing cells, e.g., cells that may be grown in a cost-efficientmanner and that may undergo programmed senescence faster than theirnormal counterparts. It will also be understood that transgenic animals(e.g., experimental and domestic animals) may be constructed of suchsmall rapidly growing cells. Skilled artisans will recognize that theadvantages offered by the subject cells include: a) improved growth intissue culture of terminally differentiated cell types that wouldnormally be difficult or impossible to culture (e.g., stem cells); andb) lessened (or no) dependence on growth factors for cell growth (e.g.,for cells that are difficult to propagate in vitro such as muscle orneural cells), allowing more rapid growth of cultures of mammalian cellsin low-serum (or serum-free) medium (e.g., in production cultures ofcells manufacturing biotherapeutic agents). The disclosure hereinidentifies the singular significance of a cyclin in determining theprogression of the cell cycle at each of several decision points. Theterm "decision point" is well accepted in the art as meaning a point inthe cell cycle where a cell may arrest its growth until such time as anappropriate signal is received to progress the cell cycle. The resultspresented in Example 14, below, illustrate the significance of cyclin Ein activating a latent CDK2 kinase activity at a decision point in theG1phase of the cell cycle. Several decision points exist during thedifferent phases of the cell cycle. The disclosure herein indicates thata small number of cyclins may be operative in the cell cycle, i.e., onecyclin to advance the cell cycle at each decision point. Thus,over-expression of a relatively small number of cyclins in a cell may besufficient to render the cell nearly completely independent of exogenousgrowth factors.

In other embodiments the invention provides immunologic binding partnersfor cyclin E polypeptides such as polyclonal and monoclonal antibodymolecules, and various antigen-binding fragments thereof, capable ofspecifically binding the cyclin E polypeptide. Such immunologic bindingpartners may be produced by hybridoma or rDNA expression techniques andfind utility in therapeutic, purification, and diagnostic applications.Therapeutic applications include binding partners that inhibit bindingof a cyclin E to its CDC protein kinase; binding partners that modulateCDC protein kinase; and binding partners that alter regulatory controlof cyclin E in a cell. Representative examples of purificationapplications include immunochemical methods and immunoaffinitychromatography. Representative examples of diagnostic applicationsinclude enzyme-linked and radioisotopic immunoassays,immunofluorescence, fluorescence immunoassay, time-resolved fluorescenceimmunoassay and the like. The specific binding partners used in theseassays may be capable of distinguishing between free cyclin Epolypeptide and cyclin E bound in complex with CDC protein kinases.Those skilled in the art will recognize that "neo" (new) antigens areacquired by conformationally altered polypeptides, and will furtherrecognize that the binding between cyclin E and a CDC protein kinaseinduces such a conformational alteration in both polypeptides. Thecyclin E-specific binding partners find general utility in diagnosticassays for detecting and quantitating levels (e.g., protein or antigen)and functional activities (e.g., phosphorylase kinase activation) offree cyclin E (or complex-associated cyclin E) in a cell such as a tumorcell. The subject diagnostic assays include assays for: a) detecting theabsolute levels and activities of cyclin E in nonsynchronized cellpopulations (e.g., in tumor biopsy specimens); b) comparing the levelsand activities of cyclin E in synchronized cell populations at differentphases of the cell cycle (e.g., cell populations synchronized bythymidine-block); and c) assays for determining the levels andactivities of cyclin E in biological fluids (i.e., blood, serum, plasma,mucus secretions, CNS fluid, cell extracts, and the like). The absolutelevels and activities of cyclin E expressed in malignant biologicalfluids (e.g., tumor cell extracts, serum from cancer patients, and thelike), as well as the levels and activities expressed in cell extractsprepared at different stages in the cell cycle, may provide informationon the rate of cell growth. In this regard the assayed levels andactivities of cyclin E may serve as diagnostic markers for:

a) staging tumors, since differentiated cells grow more slowly thantransformed; express lower levels of cyclin E protein and activity thantransformed cells; and (in contrast to transformed) differentiated cellsexpress cyclin E only during G1 phase of the cell cycle;

b) determining prognosis, i.e., predicting patient survivability andtime to recurrence of tumor, because rapidly growing malignant cellscapable of metastasis may generally grow more rapidly thandifferentiated cells; exponentially growing cells may express higherabsolute levels (or activities) of cyclin E, or alternatively, thehigher levels (or activities) of cyclin E may be present during aparticular phase of the cell cycle, e.g., during S, G1 or G2 phase;and/or

c) predicting therapeutic success, i.e., of a particular therapeuticregimen, because more slowly growing cells may express lower levels (oractivities) of cyclin E (i.e., than rapidly growing metastatic cells)and also be more responsive to less drastic and more prolongedtherapeutic regimens.

The subject assays for determining the level (or activities) of cyclin Ein cells may also indicate the responsiveness of a patient's tumor to aparticular therapeutic agent exerting its affect on cells during the G1phase of the cell cycle. Those skilled in the art will recognize thatthe subject diagnostic assays may provide results that are potentiallyuseful to a physician in deciding how to stage a tumor, how to select anappropriate therapeutic regimen, how to evaluate the success of therapy,and how to evaluate patient risk or survivability.

In other embodiments, the invention provides diagnostic assays formeasuring the absolute levels (or functional activities) of cyclin E:CDK2 polypeptide complexes in asynchronous cells, and the levels (oractivities) of the cyclin E:CDK 2 complexes at different stages in thecell cycle. In nontransformed cells the peak abundance of the cyclinE:CDK 2 complexes occurs late in the G1 phase of the cell cycle, e.g.,with levels 4-fold to 6-fold higher than in other phases of the cellcycle. In transformed cells constitutive over-expression of cyclin Eand/or CDK 2 may lead to differences in the levels of the cyclin E:CDK 2complexes that are detectably different during the G1, G2, M, and Sphase of the cell cycle. The subject assays that determine the levels ofcyclin E/CDK 2 complexes in cells may be useful in assessing malignantcells from patients, e.g., as described above.

In other embodiments the invention provides diagnostic assays fordetermining chromosomal rearrangement of cyclin E and CDK2 genes in acell. The chromosomal location of cyclin E and CDK2 genes isconveniently determined in chromosomal smears by in situ hybridizationwith oligonucleotide probes or cDNA and the like. Translocation of acyclin E gene or CDK2 gene, i.e., from a chromosomal location found in anormal cell to a location found in a transformed cell, may contribute toa phenotype of uncontrolled cell growth by removing normal transcriptionregulatory control of either gene expression of a cyclin, e.g., cyclinE, or a CDC kinase, e.g., CDK2. The findings disclosed herein indicateheretofor unrecognized common junction points where second messengersignals from multiple growth factors converge at a small number ofdifferent cyclin:CDC kinase complexes. The outcome of the molecularinteraction of the second messengers with the cyclin:CDC kinasecomplexes determines whether the cell progresses the cell cycle. Thus,rearrangement of a cyclin gene in a cell may have dramatic results. Inthe case where the rearrangement induces over-expression the cell mayacquire a malignant (i.e., uncontrolled) growth phenotype, and in thecase where the rearrangement induces under-expression the cell mayundergo premature senescence. Screening cellular samples fromindividuals for the potential of cyclin E or CDK2 chromosomalrearrangement may indicate a relative risk factor for the possibility ofdeveloping cancer. In the event that such rearrangements are detected,restoring normal control of a cyclin gene (e.g., cyclin E) or a CDCkinase gene (e.g., CDK2) in a translocated chromosomal location mayreverse the malignant phenotype of a transformed cell. For example, thecyclin E gene (of CDK2 gene) and its regulatory elements may serve astargets for gene therapy vectors designed to inactivate the rearrangedgene, e.g., using in situ-directed recombination/mutagenesis or targetedintegration to disrupt the translocated gene.

The invention also provides transgenic strains of yeast cells that areengineered to contain a genome lacking in cln1, cln2, and cln3 genesrequired for cell cycle progression but having an episomal nucleic acidcapable of encoding CLN3. A representative embodiment is yeast strain"589-5", which is useful to identify mammalian cyclin genes. Strain"589-5" has been deposited with the American Type Culture Collection,Rockville, Md., under accession No. 74098. When cyclin E nucleic acid isintroduced into the transgenic yeast cells of this strain, the cellsadvance through the cell cycle from START into S phase. While othershave shown previously that somewhat similar transgenic yeastconstructions are useful for identifying and cloning yeast cdc genes,the subject strains are useful for identifying and cloning mammaliancyclin E cDNA, and also for identifying and cloning mammalian cdcprotein kinase cDNAs.

The invention also provides other transgenic strains of yeast cell,having a cdc28-13 gene, an endogenous GI CLN (e.g., CLN3) under controlof a conditional promoter (e.g., GAL), a mitotic CLB cyclin (e.g., CBC),and a cyclin E gene, such as the representative yeast strain"1238-14C-cyclin E". In this case the conditional promoter drivesexpression of the G1 cyclin in the presence of a factor ("thecondition") required for metabolism or growth. The G1 cyclin, in turn,binds and activates the cell division kinase encoded by the cdc28-13gene, and activation of the CDK allows the cells to be grown andpassaged. Strain "1238-14C-cyclin E" has been deposited with theAmerican Type Culture Collection, Rockville, Md., under accession No.74099. The cell cycle is blocked in these cells until a cdc proteinkinase nucleic acid is introduced. Thus, this strain of transgenic cellsis useful for identifying and cloning cdc protein kinases that associatewith a cyclin E and advance the cell cycle from G1 into S phase in aeukaryotic host cell.

The invention also provides methods for cloning regulatory agents suchas polypeptides that bind to cyclin E:CDC protein kinase complexes andinhibit or promote the activity of the complex, or that change thehalf-life of the complex, and the like. Nucleic acids encoding suchregulatory agents are identified by introducing a candidate nucleic acidmolecule into a transgenic strain of yeast cell, or a mammalian cell, inwhich advance of the cell from G1 into S phase is dependent upon theactivity of a cyclin E:CDC protein kinase. A representative example of atransgenic yeast strain useful in this manner is provided by strain598-5 transfected or transduced with a cyclin E gene, such as strain HU4described in greater detail in the Examples below. Nucleic acidsencoding regulatory agents are recognized by their ability to inhibitprogression of the cell from G1 into S. Thus, in the case of thedescribed transgenic yeast strain, replicative screening techniques maybe used for identifying clones of cells that fail to advance the cellcycle at START after they have been transfected or transduced with anycandidate cDNA, e.g., mammalian, insect, avian, reptilian, amphibian,etc.

EXAMPLES

The data set forth below show that cyclin E substituted for the S.cerevisae CLN genes and interacted with CDC28 to perform START. At leasttwo different members of the human CDC2 gene family could interact withcyclin E to regulate START in budding yeast, CDC2-HS and CDK2-HS. Wehave also shown that the cyclin E protein bound and activated the p34CDC2 protein in extracts from human lymphoid G1 cells and that cyclin Ewas associated with an H1 kinase activity in HeLa cells. Others foundthat the cyclin E mRNA was specifically present during the late G1 phaseof the HeLa cell cycle (Lew et al., 1991). Taken together these resultsargue that cyclin E may function as a regulator of the p34 CDC2 kinaseat the G1 to S transition in the human cell cycle.

The interpretation of our result that cyclin E rescued the triple clndeletion was complicated by the fact that human cyclin B, which is amitotic cyclin, also substituted for the yeast CLN genes. Regardless ofthe mechanism by which cyclin B functioned as a CLN protein, the factthat it played this role implied that our complementation assay did notspecifically identify CLN-type cyclins. Therefore, a more completeunderstanding of the function of cyclin E must await analyses of theabundance of cyclin E protein and its association with CDC2, orCDC2-related proteins, during the cell cycle of normal human cells.

In yeast, and probably in most organisms, the CDC2 protein functions atleast twice during the cell cycle: at G1/S and again at G2/M. At eachpoint the CDC2 protein associates with a unique type of cyclin: theB-type cyclins at G2/M and the CLNs (at least in budding yeast) at G1/S.Therefore, it had been expected that the unique cyclin-CDC2 complexwhich assembled at each control point would impart the specificityrequired for the CDC2 kinase to activate either S phase or mitoticevents. For example, the substrate specificity or subcellularlocalization of the CDC2 kinase would be determined by its particularcyclin partner. The ability of human cyclin B to substitute for the CLNproteins appears, on the surface, to contradict this simple hypothesis.As an alternative it is possible that the specificity of p34 CDC2 actionat different points in the cell cycle might be determined, at least inpart, by the CDC2 protein itself. This could be due to cellcycle-specific modification of the CDC2 protein (Simanis & Nurse, 1986;Lee et al., 1988; Gould & Nurse, 1989; Krek & Nigg, 1991) or, in highereukaryotes, to the activation of different members of the CDC2 genefamily at different points in the cell cycle (Paris et al., 1991; Pines& Hunter, 1990). Our observation that at least two different members ofthe CDC2 gene family can perform START in yeast is consistent with thisidea. In this model periodic accumulation and destruction of the cyclinproteins would determine the timing of p34 kinase activation but not itsspecificity. A model similar to this has been proposed previously basedupon the phenotypes of certain cdc2 mutants in S. pombe (Broek et al.,1991). An alternative is that the particular substrates necessary forCDC2 or CDC28 to induce the S or M state become accessible in a cellcycle-dependent manner. We point out, however, that the design of ourexperiments may not have permitted all the normal controls on cyclinspecificity to be observed. For example, expression of human cyclin Bfrom the strong ADH promoter might have overwhelmed certain regulatoryprocesses that usually limit cyclin B-CDC28 activity to the G2/Mtransition.

Human cyclin E is more closely related to human cyclins A and B than tothe budding yeast CLN proteins. Within the cyclin box the level ofidentity to CLN1 is 21% and to CLN3, 17%. This compares to 49% identityto human cyclin A and 44% to human cyclin B. Outside the cyclin boxregion cyclin E shows no extensive homology to either the human cyclinsor yeast CLN proteins. On this basis cyclin E does not appear to be adirect homolog of the yeast CLN genes. This comparison must be made withcaution, however, since the precise regions within the various cyclinsthat determine their functional differences have not been identified. Inaddition, the similarity among the human cyclins may reflect, to someextent, their co-evolution with common targets such as the human CDC2protein.

Two other features of the cyclin E sequence should be noted. Our cloneof cyclin E lacks an N-terminal sequence found in both cyclin A and B,which is thought to be a recognition motif for the ubiquitination enzymethat mediates their destruction in mitosis (Glotzer et al., 1991).Furthermore, cyclin E contains a C-terminal extension when compared tocyclins A and B. This C-terminal region is flanked by basic residues andis rich in P (Pro), E (Glu), S (Ser), and T (Thr) residues. Such "PEST"regions have been implicated in controlling protein turnover (Rogers etal., 1986). In fact, the stability of the yeast CLN proteins may bedetermined by their C-terminal domains which are also rich in P, E, S,and T residues (Nash et al., 1988; Cross, 1990; Hadwiger et al., 1989).These observations suggest that the stability of cyclin E during thecell cycle might be controlled differently than the mitotic cyclins.

In higher eukaryotes the role of the CDC2 protein during progressionthrough the G1 phase of the cell cycle is not well understood. A numberof independent observations suggest that the CDC2 protein has anessential function during this part of the cell cycle. Depletion of theCDC2 protein in vivo in human cells, using antisense oligonucleotides(Furakawa et al., 1990), or in vitro in Xenopus extracts, byimmunoprecipitation (Blow & Nurse, 1990), can block the start of DNAsynthesis. Addition of cyclin-CDC2 complexes from human S phase cells toextracts from G1 cells can activate DNA synthesis (D'Urso et al., 1990).However, a thermolabile mutation of the murine CDC2 gene blocks entryinto mitosis but not S phase at the nonpermissive temperature (Th'ng etal., 1990). Also, microinjection of antibodies against the yeast CDC2protein into human cells blocks the G2/M but not the G1/S transition(Riabowol et al., 1989). Our observation that at least two differentmembers of the CDC2 gene family can regulate the G1/S transition in S.cerevisae may help clarify these apparently contradictory results. Oneof these, the human CDK2 gene, might preferentially work at G1/S asopposed to G2/M. Therefore, in higher eukaryotes, in contrast to thesituation in yeast, multiple members of the CDC2 gene family mayparticipate in G1/S regulation. Under some circumstances their rolesmight be redundant, while in other situations they may all be essential.

Activation of the CDC2 kinase during the G1 to S interval is likely torequire its association with a cyclin. In S. cerevisae accumulation of aCLN-type cyclin is the rate-limiting step for transit through START(Nash et al., 1988; Cross, 1988; Hadwiger et al., 1989). In human cellsactivation of the p34 kinase at the start of S phase correlates with itsassembly into a higher molecular weight complex (D'Urso et al., 1990;Marraccino et al., unpublished data), implying that association of p34with a cyclin protein regulates its activity during this part of thecell cycle. We have also found that addition of purified recombinantclam cyclin A to a human G1 cell extract was sufficient to activate SV40DNA replication, suggesting that accumulation of a cyclin is thelimiting step for activation of the p34 kinase at the start of S phase.

At least four different human cyclins have been suggested to play rolesduring the G1 or S phases of the cell cycle. These include cyclins A(Giordano et al., 1989; Wang et al., 1990), C (Lew et al., 1991), D (orprad1; Motokura et al., 1991; Xiong et al., 1991; Matsushimi et al.,1991), and E (this disclosure; Lew et al., 1991). In yeast, human cyclinE can associate with either CDC2-HS or CDK2-HS to perform START. Invitro cyclin E can bind and activate CDC2-HS, but its ability toactivate CDK2 from G1 cells has not yet been tested. Human cyclin A hasalso been found to associate with two different members of the CDC2 genefamily, CDC2-HS and a 33 kDa protein which may be CDK2 (Giordano et al.,1989; Pines & Hunter, 1990; R. Marraccino & J. R., unpublishedobservations). In contrast, the mitotic cyclin B has been found inassociation only with p34 CDC2 (see Hunt, 1989).

Multiple cyclins and CDC2-like proteins may be required to convey thediverse array of intracellular and extracellular signals that contributeto G1 regulation. Different members of the CDC2 protein family maypreferentially interact with particular cyclins (Pines & Hunter, 1990).Also, each cyclin-CDC2 complex may perform only a subset of the eventsnecessary for START to occur. Finally, it is possible that the CDC2family of proteins may function at more than one point during G1. TheSTART decision in yeast is the only clearly defined execution point forCDC28 during the G1 phase of the cell cycle. START bears certainsimilarities to the restriction point in the cell cycle of highereukaryotes; however, the restriction point can occur hours before thestart of S phase. Our in vitro replication experiments indicate that theCDC2 kinase may directly activate DNA synthesis (D'Urso et al., 1990).Therefore, CDC2, or related proteins, may function twice during G1,first at the point of commitment to cell proliferation and again at theonset of DNA synthesis. Each control point may require a unique set ofcyclin proteins; for example the CLN-type cyclins may function at therestriction point and other cyclins, such as cyclin E, could act at theG1/S transition.

EXAMPLE 1 Complementation of a Yeast Strain Lacking CLN1, -2 and -3 witha Human cDNA Library

Our initial goal was to identify human cDNAs encoding proteins thatcould substitute for the yeast CLN proteins. A yeast strain, 589-5, wasconstructed in which all three chromosomal CLN genes were deleted andwhich contained the CLN3 gene under the control of the GAL1 promoter onan episome. As CLN protein is required for passage through START, thisstrain will grow on galactose, where the GAL1 promoter is induced; whenthis strain is grown on glucose the GAL1 promoter is repressed, no CLN3protein is made, and the cells arrest at START (Cross, 1990). A cDNAlibrary (a gift of J. Colicelli and M. Wigler), using mRNA prepared fromthe human glioblastoma cell line U118, was constructed in a S. cerevisaevector containing the constitutive yeast ADH promoter for expression ofthe human cDNAs (Colicelli et al., 1989). The library was transfectedinto strain 589-5, and 10⁵ independent transformants were screened, byreplica plating, for their ability to grow on glucose. One transformant,HU4, was isolated whose growth on glucose was dependent upon expressionof a human cDNA (FIG. 1). For further experimental details, see theappended Materials and Methods.

EXAMPLE 2 HU4 Encodes a New Member of the Cyclin Protein Family

The DNA sequence of the 1.7 kb HU4 cDNA is shown in FIG. 2, and itshomology, at the protein level, to human cyclins A and B is illustratedin FIGS. 3A-3B. The DNA sequence predicts a protein with 395 amino acidsand a molecular weight of 45,120 daltons. In vitrotranscription/translation of the HU4 cDNA yields a protein with thepredicted molecular weight (see FIGS. 8A-8B). All known cyclins have ahighly conserved central domain of approximately 87 amino acids. HU4 is49% identical to human cyclin A and 44% identical to human cyclin Bwithin this domain. On this basis we placed the HU4 protein within thecyclin family. N-terminal to this conserved region the homology tocyclin A falls to 5% identity, and 4% identity to cyclin B. C-terminalto this domain the identity to cyclin A is 14%, and the identity tocyclin B is 10%. This low level of homology both N- and C-terminal tothe central conserved domain suggests that HU4 represents a new class ofcyclin proteins, and we have designated this class as cyclin E. Wecannot be certain that our cyclin E cDNA clone contains the entirecyclin E protein coding sequence, as the open reading frame extends tothe 5' end of the sequenced cDNA. However, a cyclin E cDNA cloneobtained from a MANCA cell library showed a 5' end identical to the onedescribed here.

EXAMPLE 3 Human Cyclin B Complements the Triple cln Deletion

We compared the ability of human cyclin A, B, and E cDNAs to complementthe triple cln deletion in strain 589-5. Full-length cDNA clonesencoding human cyclins A (Pines & Hunter, 1990) and B1 (Pines & Hunter,1989) were cloned into the ADH expression vector used in construction ofthe library described above. The various cyclin E expression plasmidswere transfected into yeast by selecting for leucine prototrophy. Thetransformants were picked, and the ability of the human cyclin tocomplement the absence of the CLN proteins tested by growth on glucose.No leucine prototrophs were obtained using the cyclin A vector,suggesting that expression of full-length human cyclin A in S. cerevisaewas lethal. FIG. 4 depicts the relative plating efficiency of 589-5 onglucose versus galactose when containing either cyclin B or E expressionplasmids. Surprisingly the mitotic cyclin B complemented the absence ofthe CLN proteins. This experiment demonstrated that complementation ofCLN function was not restricted to CLN type cyclins.

EXAMPLE 4 Interaction between Cyclin E and CDC28

We compared the ability of cyclin E to rescue the triple cln deletion inisogenic strains containing either the CDC28 or the cdc28-13 allele atthe permissive temperature of 30° C. FIG. 4 shows that cyclin Esubstituted for the CLN proteins significantly less well in the cdc28-13background. This genetic interaction between the cyclin E and CDC28genes suggested that the cyclin E protein performed its function byinteracting with the CDC28 protein. Cyclin B was about as effective incdc28-13 versus CDC28 strains, suggesting that cyclin B might interactwith CDC28 differently than cyclin E.

EXAMPLE 5 Human Cyclin E and Human CDC2 Can Perform START in S.cerevisae

Strains containing cdc28-13 and the endogenous G1 (CLN) and mitotic(CLB) cyclins are viable at 30° C. Therefore, the cdc28-13 protein mustbe capable of functional interactions with both types of cyclins. Asdescribed above, a cdc28-13 strain that contained cyclin E in place ofthe CLN genes did not grow at 30° C. We speculated that this strain wasdefective specifically for the START function of the CDC28 protein andnot for its G2,/M role, presumably because the cdc28-13 mutationdiminished its ability to interact productively with cyclin E. Theseproperties of the cyclin E-cdc28-13 strain are shown in FIG. 5. We usedthis strain as a host to screen for human genes that could interact withcyclin E to perform START. Unlike screens requiring human genes tocompletely substitute for CDC28, this screen may not require that thehuman genes function at G2/M. We transfected this strain with the humancDNA expression library described above, and 10⁵ independent colonieswere tested for their ability to grow on glucose. We identified fiveyeast clones whose growth depended upon expression of both the humancDNAs (cyclin E and the new one) (FIG. 6). The human cDNA within each ofthese clones was restriction mapped (data not shown). Four of these(S2-6a2, S2-103, S2-112, S2-227) contained the CDC2-HS gene (Lee &Nurse, 1987). The S. pombe cdc⁺ gene also performed START together withhuman cyclin E in this strain (F. C. & A. Tinkelenberg, unpublishedobservations). These results provide further evidence that the cyclin Eprotein controls START through its interaction with the CDC2 or CDC28protein. The fact that the S. cerevisae CDC28 and CLN genes can bereplaced simultaneously by human proteins also emphasizes the extent towhich the basic cell cycle machinery has been conserved in evolution.

EXAMPLE 6 Transit through START in Yeast Containing Human Cyclin E andHuman or Xenopus CDK2, a Member of the CDC2 Gene Family

The restriction map of the fifth clone, S2-124, did not match that ofCD2-HS. Recently, the human homolog of the Xenopus CDK2 gene (formerlycalled Eg-1; Paris et al., 1991) was cloned (S. Elledge, personalcommunication), and the restriction map of S2-124 matched that ofCDK2-HS. In addition we found that the Xenopus CDK2 gene alsosubstituted for CDC28 and performed START in conjunction with cyclin E.Therefore, humans and Xenopus contain at least two members of the CDC2gene family that can control the G1/S transition in yeast. In order totest whether the human CDC2 gene, or the CDC2 homolog CDK2, fullycomplemented cdc28-13, we grew these transformants at 38° C. (FIG. 5).At 38° C. cdc28-13 is defective compared to wild-type CDC28 for bothG1/S and G2/M (Reed & Wittenberg, 1990). As expected (Wittenberg & Reed,1989) cells containing the CDC2-HS gene grew equally well at 30° or 38°C., showing that the CDC2-HS gene was able to complement both the G1/Sand G2/M functions of cdc28-13. Curiously, at 38° C. the strainscontaining the human or Xenopus CDK2 gene and human cyclin E failed togrow. Therefore, under these experimental conditions, the human orXenopus CDK2 genes only partially substituted for CDC28. In addition,initial attempts to complement a GAL-CDC28 strain with CDK2-HS (onglucose) showed that complementation was extremely poor compared withcomplementation by CDC2-HS. The failure of CDK2-HS to fully complementeither cdc28-13 or GAL-CDC28 is consistent with a previous reportshowing that the Xenopus CDK2 gene did not complement either cdc28 orcdc2 temperature-sensitive alleles (Paris et al., 1991). The explanationfor the partial rescue of cdc28-13 by CDK2 is unclear, but onepossibility is that the CDK2 protein can complement effectively the G1/Sbut not the G2/M function of CDC28. To address this issue definitively,it would be essential to determine the cell cycle arrest points of thestrains described above. This has not been possible because theinstability of the plasmids resulted in only a minority of the cellscontaining all three plasmids even on selective medium (data not shown).

EXAMPLE 7 Activation of Human p34 CDC2 by Cyclin E

Mixing cyclin E protein with G1 cell extracts demonstrated directly thatcyclin E could bind the human CDC2 protein in vitro and that thisassociation led to activation of the CDC2 kinase. We have shownpreviously that human G1 cells contain no active p34 CDC2 kinase; allthe P34 protein present in the cell is monomeric, unassociated with anycyclin (Draetta & Beach, 1988; D'Urso et al., 1990). G1 extracts wereprepared from MANCA cells, a human Burkitt's lymphoma cell line. Weconfirmed that these G1 extracts contained no detectable CDC2 kinase.The extracts were mixed with a vast excess of p13-Sepharose relative toCDC2 protein, conditions that ensure quantitative binding of CDC2protein to p13-Sepharose. No histone H1 kinase activity could bedetected specifically associated with the p13-Sepharose beads (see FIG.7B). Also, these G1 extracts were inactive in a kinase assay using aspecific peptide substrate of the CDC2 kinase (Marshak et al., 1991).

To study the interaction between cyclin E and CDC2, cyclin E wasexpressed in E. coli as a glutathione transferase fusion protein(GT-cyclin E) and purified by affinity chromatography onglutathione-Sepharose. We incubated the G1 cell extract with GT-cyclinE-Sepharose, GT-Sepharose, p13-Sepharose, and blank Sepharose beads.GT-cyclin E-Sepharose and p13-Sepharose bound equivalent amounts of p34CDC2 protein, as detected by immunoblotting using a C-terminus-specificp34 CDC2 antiserum (FIG. 7A). We detected no binding of p34 CDC2 toGT-Sepharose or blank Sepharose.

After incubation in the G1 extract the Sepharose beads were assayed forhistone H1 kinase activity. Only the GT-cyclin E-Sepharose beadscontained histone H1 kinase activity, even though the p13-Sepharose andGT-cyclin E-Sepharose beads bound equal amounts of p34 CDC2 protein(FIG. 7B). We also observed that the cyclin E fusion protein wasphosphorylated by the bound kinase. A protein precisely comigrating withthe cyclin E fusion protein was phosphorylated during the H1 kinasereaction, and this phosphoprotein was immunoprecipitated by a cyclin Eantiserum (FIG. 7B). Cleavage of the phosphorylated GT-cyclin E fusionprotein with thrombin showed that the cyclin E portion of the fusionprotein was phosphorylated (data not shown). Phosphorylation of theGT-cyclin E fusion protein was probably due to the bound CDC2 kinase(see below), since autophosphorylation of the cyclin subunit ischaracteristic of cyclin-CDC2 complexes (Draetta & Beach, 1988; Pines &Hunter, 1989).

The experiments described above did not demonstrate directly that theGT-cyclin E-associated kinase was the p34 CDC2 kinase. To test this, wereleased the GT-cyclin E-associated proteins from the Sepharose beads byincubation with free glutathione. The released p34 CDC2 protein wasimmunoprecipitated with a C-terminus-specific p34 CDC2 antiserum andshown to have histone H1 kinase activity (FIG. 7C). As a control weshowed that no kinase was immunoprecipitated from the protein releasedfrom the GT-Sepharose beads (FIG. 7C). We do not know what fraction ofthe GT-cyclin E-bound kinase could be immunoprecipitated with the p34CDC2 antiserum and therefore cannot rule out that other kinases (such asCDK2) contributed to the GT-cyclin E-bound kinase activity.

Our results show that cyclin E bound the p34 CDC2 kinase and support theidea that it was activated by cyclin E. The fact that no CDC2 kinase wasdetected in the initial G1 extract suggests that association of the CDC2protein with the GT-cyclin E-Sepharose led to activation of previouslyinactive protein. Since these experiments were done in crude cellextracts, they could not address whether the association of cyclin Ewith CDC2 protein was sufficient for activation of the CDC2 kinase.Additional modifications of either the CDC2 or cyclin protein may benecessary steps in the activation pathway.

Antibodies were raised in rabbits against the GT-cyclin E fusionprotein. These antibodies specifically recognized cyclin E, as theyimmunoprecipitated in vitro translated cyclin E, but not human cyclin Aor B (FIG. 8A). This antiserum immunoprecipitated an H1 kinase activityfrom HeLa cells (FIG. 8B). This suggested that cyclin E associated witha kinase in vivo, although we do not know which members of the CDC2family were present in these complexes.

EXAMPLE 8 Cell Cycle Dependent Activation of Cyclin E and CyclinA-associated Protein Kinases

The previous Examples show that immunoprecipitates of cyclin E fromexponentially growing MANCA cells (a human B cell line) contain a celldivision kinase. Cyclin E-associated kinase activity during the cellcycle was investigated using centrifugal elutriation to separateexponentially growing MANCA cells into 8 fractions. Centrifugalelutriation physically separates cells into different cell cyclefractions thereby avoiding potential artifacts known to be associatedwith induced synchronization procedures. We determined the position ofan elutriated fraction by measuring the nuclear DNA content by flowcytometric analysis of propidium iodide stained nuclei (FIG. 9A).Cellular extracts from each of the eight different cell cycle fractionswere immunoprecipitated using an anti-cyclin E polyclonal antiserum.Pre-immune serum (αPI) from the same animal was used as a negativecontrol. For each fraction, the kinase activity in the controlimmunoprecipitates was subtracted from the activity observed in thespecific anti-cyclin E immunoprecipitates. The data, presented in FIG.9B, show the level of expression of cyclin E-kinase complexes in theelutriated fractions of cells (FIG. 9A) as determined by measuring thelevel of histone H1 phosphorylation catalyzed by cyclin E-associated H1kinase activity. Equal numbers of cells from each fraction were lysed,and cyclin E in the lysates was immunoprecipitated with affinitypurified anti-cyclin E antibodies (αcycE). The results, quantitated byphosphor-imaging, indicate that cyclin E-associated kinase activity wascell-cycle-dependent and, in three experiments, fluctuated during thecell cycle by between 4- and 8-fold. The peak in cyclin E-associatedkinase activity corresponded to elutriated fractions of cells having thegreatest number of cells in late G1 and early S phase. In someexperiments, we also observed a smaller second peak of cyclinE-associated kinase activity in the G2/M fraction of elutriated cells(data not shown).

The activity of the cyclin E-associated kinase during the cell cycle ismarkedly different from the kinases associated with cyclin A. C160anti-cyclin A monclonal antibodies were used to immunoprecipitate cyclinA and its associated proteins from the same cell extracts that had beenused to measure cyclin E-associated kinase activity (FIG. 9C). Aspreviously described, cyclin A-associated kinase activity is firstdetected at the start of S phase (Pines and Hunter, 1990; Marraccino etal., 1992). In contrast to cyclin E-associated kinase activity, thecyclin A-associated kinase activity continues to rise throughout S phaseand peaks in G2. These results also indicate that peak levels of thecyclin A-associated kinase are approximately 5 to 10-fold greater thanthe peak activity levels of the cyclin E-associated kinase (data notshown). However, the absolute levels may vary since the levels presentedhere depend upon two antibodies that may have slightly differentassociation constants (K_(a)). These results suggest that a successionof distinct cyclin dependent kinase activities are activated during thecell cycle; kinase activity of cyclin E increases, followed by anincrease in cyclin A- then cyclin B-associated kinase activity.

The kinetics with which the cyclin E-associated kinase activityaccumulate during the G1 phase of the cell cycle was investigated as arelative measure of the abundance of the enzymatically active cyclinE:kinase complex. MANCA cells were arrested for 3 hours in the metaphasestage of the cell cycle with nocodazole at which time 75% of the cellshad completed cytokinesis. Cells seperated from residual mitotic cellsby elutriation were then released into the G1 phase of the cell cyclefor 3,4,5,6 or 7 hours. They progressed synchronously into S phase afterabout 6 or 7 hours as determined by both flow cytometric measurement ofnuclear DNA content (9D) and tritiated thymidine incorporation intochromosomal DNA (data not shown). Cells were then fractionated intosub-populations in different phases of the cell cycle by centrifugalelutriation. Cyclin E-associated kinase activity was found to beelevated during the G1 period reaching peak activity just as the cellsentered S phase (FIG. 9D). In contrast, we found that cyclinA-associated kinases were not present in G1 and were first detected ascells entered S phase (FIG. 9E). In this experiment, cyclin A-associatedH1 kinase activity was determined using C160 anti-cyclin A monoclonalantibodies to immunoprecipitate cyclin A-associated kinase activity. Theelutriated G1 fraction of cells (fraction 2) was cultured at 32.5° C. toexpand the G1 phase of the cell cycle. Aliquots of cells were harvestedhourly for measurement of nuclear DNA content, and cyclin A- and cyclinE-associated kinase activities, up to the point where the cellsapproached and entered S phase.

Cyclin E-associated kinase is readily detectable in proliferating rat208F cells but disappears when they enter quiescence after serumwithdrawal (FIG. 10A). Similarly, when rat PC 12 cells were induced todifferentiate into neurons by exposure to NGF, the cyclin E-associatedkinase fell to low levels (FIG. 10B). In these experiments, we assayedH1 kinase activity in lysates from growing and quiescent rat 208F cells,and growing rat PC-12 cells. Immunoprecipates were prepared usingaffinity-purified antibodies against cyclin E (αE), the C-terminus ofhuman p34 CDC2 (αp34), or as a control pre-immune anti-cyclin Eantiserum (αPI). Cells were induced to grow using 10% calf serum (10%CS, FIG. 10A) and to differentiate using NGF (+NGF, FIG. 10B). Quiescentcontrols were grown in 0.1% calf serum (0.1% CS, FIG. 10A).Nondifferentiating controls were grown in the absence of NGF (-NGF, FIG.10B). These results demonstrate that cyclin E-associated kinase activityis growth regulated as are the levels of cyclin E expression.

EXAMPLE 9 Constitutive Expression of Cyclin E Shortens G1

The pattern of cyclin E-associated kinase activity during the cell cyclesuggested that the physiological function mediated by cyclin E takesplace during the G1 phase of the cell cycle. To test this possibility,stable cell lines were constructed that constitutively expressed humancyclin E from a retroviral LTR promoter. The effects of constitutivecyclin E expression on cells, and on cell cycle kinetics was tested.

A human cyclin E cDNA was cloned into the retroviral expression vector,LXSN (Miller & Rosman, 1989), which expresses the inserted cDNA from the5'LTR and contains the neomycin phosphotransferase gene as a selectablemarker. The construct permits production of retrovital vector particleshaving either amphotropic or ecotropic host range specificity. We usedan ecotropic stock of vector particles to infect the Rat-1 fibroblastcell line and selected a pool of over ten thousand independentlyinfected cells by growth for 2 weeks in G-418 containing selectivemedia. At the same time, a pool of LXSN control vector-infected cellswas generated. Pools of infected cells were studied rather thanindividual selected cell clones to minimize any possible clonalvariation that might be present within the Rat-1 cell line. The results,presented in FIG. 11A, show that the LXSN-cyclin E infected cellscontained approximately 5 to 10-fold more cyclin E protein than could bedetected in the cells infected with the control LXSN viral vectors. Twocyclin E bands, at 45 kDa (the size of full length cyclin E) and 40 kDa,were specifically expressed in cyclin E transduced cells. Increasedcyclin E:kinase activity was also observed in the LXSN-cyclin Etransduced cells. The results in FIG. 11B show a 3 to 5-fold increase inthe level of cyclin E-associated histone H1 kinase activity inexponentially growing cultures of Rat-1 cells. The lower levels ofcyclin E and cyclin E-associated kinase detected in the control cellswas presumably due to endogenous rat cyclin E. To evaluate the effectsof cyclin E on the cell cycle, exponentially growing LXSN-cyclin Etransduced cells were collected by centrifugal elutriation. Thedistribution of transduced cells within the cell cycle was determined byflow cytometry after DNA staining with propidium iodide (FIG. 11C).Cells transduced with LXSN-cyclin E showed a decrease in the fraction ofcells in the G1 phase of the cell cycle in comparison to control cellstransduced with the control vector, LXSN. The cyclin E transduced cellsalso showed an increase in the fraction of cells in the S phase of thecell cycle. These observed changes in the fractions of cyclin Etransduced cells in the different phases of the cell cycle areconsistent with accelerated transit of the cells through the G1 phase ofthe cell cycle. This was confirmed by directly measuring the length ofthe G1 phase in cyclin E infected cells. Cyclin E infected cells weresynchronized in pseudometaphase by exposure to nocodazole, and mitoticcells collected. The mitotic cells were returned to culture and entryinto S phase was monitored by pulse labeling with BrdU(5-bromodeoxyuridine). The BrdU was detected using immunochemicalmethods. The results, presented in FIG. 11D, show that the length of theG1 phase in the LXSN-cyclin E infected cells (i.e., from conclusion ofmitosis until resumption of DNA synthesis in S-phase) was substantiallyshorter than in cells transduced with LXSN.

In 5 separate experiments, using two independent pairs of transfectedcell populations, the duration of G1 was, on average, 33% shorter incells infected with the LXSN-cyclin E retrovital vector than in cellsinfected with the LXSN control vector. Similarly, studies conducted tomeasure the rate of entry of cyclin E-transduced cells into S phaseusing immunochemical detection of BUDR incorporated into nuclear DNAconfirmed shortening of the S phase in LXSN-cyclin E transduced cells(data not shown). Due to the limited number of cells that can beobtained by mitotic shake-off methods, it was not possible to measurethe level of cyclin E-associated kinase activity at each time pointduring progression from mitosis to S phase in the infected Rat-1 cellpopulations.

EXAMPLE 10 Cyclin E-associated Proteins

Previous Examples have shown that cyclin E can activate human p34 CDC2,and human or Xenopus p33 CDK2, when the proteins are expressed togetherin budding yeast. Furthermore, the results have shown that cyclin E canbind and activate both human CDC2 and human CDK2 in vitro (i.e., incell-free systems). The association of kinases with cyclin E wasexamined in in vivo studies, (i.e., in cells), where cell extracts wereprepared from elutriated cell cycle fractions of exponentially growingMANCA cells that were biosynthetically radiolabeled with [³⁵S]-methionine for 3 hours. Extracts were prepared in SDS-RIPA buffer andthe specific proteins were immunoprecipitated using affinity purifiedanti-cyclin E antibodies (prepared against GST-cyclin E fusion protein),and an antiserum against the C-terminus of human CDC2 (p34).Immunoprecipitates were collected, washed, and boiled in the SDS bufferprior to separation on 12% SDS-polyacrylamide gels. Detection ofradiolabeled polypeptides in the gels was facilitated using sodiumsalicylate; and the gels were then dried for autoradiography. FIG. 12shows the p34-associated proteins (Lane 1) and the proteins associatedwith cyclin E (Lanes 2-11) in exponentially growing cells (Lane 2),during G1-phase (Lane 3), G1/S (Lane 4), S (Lanes 5-8), S/G2 (Lanes9-10) and M-phase (Lane 11). The molecular weights of the proteinsassociating with the cyclin E:CDC kinase complexes in the assay of FIG.12 are summarized in Table 1, below. The 13 Kd polypeptide wasassociated with the complex during the middle of S-phase; the 17 Kd,throughout the cell cycle; the 32 Kd-doublet, mostly late in G1 and S;the 36 Kd, only in G1 and G2/M; the 70 Kd, predominantly in S; the 85Kd, (i.e., the lowest band in the triplet) throughout the cell cycle;and, the 107 Kd, in S-phase and just before and after S-phase. Thedifference in the expression pattern of the 32 Kd-double suggested thatit was not CDK2 or CDC2, and this was confirmed by mapping the trypticfragments of CDK2, CDC2, and the 32 kD band, designated band "x",associated with the immunoprecipitated complexes.

                                      TABLE 1                                     __________________________________________________________________________    Molecular Sizesof Cyclin E:CDC kinase Accessory Proteins                                     Presence of Band with Apparent                                         Cell Cycle                                                                           Molecular Size (Kd).sup.c                                      Immunoppt..sup.a                                                                      Phase.sup.b                                                                          13  17 32 36  70 85 107                                        __________________________________________________________________________    anti-p34                                                                              Exponential                                                                          +/- +  -  +/- -  -  -                                          anti-cyclin E                                                                         Exponential                                                                          +/- +  2+ +/- 2+ +  +/-                                                G1     +   +  +  +   +  +  -                                                  G1/S   +   +  2+ +/- 2+ +  +                                                  S      2+  +  3+ +/- 3+ 3+ +                                                  S/G2   2+  2+ 3+ +/- 2+ 3+ +                                                  G2/M   +   2+ 3+ 2+  +  3+ +/-                                        __________________________________________________________________________     a.) Immunoppt. = immunoprecipitate prepared with antip34 CDC2 or              anticyclin E;                                                                 b.) Cell cycle phase, centrifugal elutriation fractions of cells; and,        c.) Molecular size in kilodaltons of polypeptides associated with cyclin      E:CDC kinase complexes, 32 kd = middle of doublet, 85 kd = lowest band of     triplet; amount indicated on a scale from - to 3+.                       

A series of control immunoprecipitaton reactions were conducted tocharacterize the specificity of anti-CDC2 and anti-CDK2 antibodies.Lysates from exponentially growing MANCA cells were immunoblotted usingaffinity purified antibodies directed toward the 7 C-terminal aminoacids of human p34 CDC2 (or CDC2) or antiserum directed toward the 15C-terminal amino acids of human p33 CDK2 (αCDK2). In FIG. 13, lanes 1and 2 are immunoblots of whole cell extracts; in lanes 3 and 4, wholecell extracts were first immunoprecipitated with affinity purifiedanti-p34 CDC2 antibodies and then blotted with the indicated antibodies;in lanes 5 and 6, extracts were first immunoprecipitated with anantiserum against the C-terminus of p33 CDK2 and then blotted with theindicated antibodies. Note the presence of a non-specific signal derivedfrom the immunoprecipitating antibody between 50 and 80 kDa. An extractfrom MANCA cells arrested at the G1/S boundary with aphidicolin wasimmunoprecipitated using affinity purified antibodies against humancyclin E and then blotted using the same antibodies. A single proteinband at 45 kDa was detected. Therefore, the associated proteins incyclin E immunoprecipitates were most likely bound to cyclin E and werenot detected due to nonspecific cross-reactivity with this antibody.

To confirm these results, immunoblotting was used to examine theassociation between cyclin E and both p33 CDK2 and p34 CDC2 in extractsprepared from MANCA cells growing exponentially or arrested at the G1/Sboundary with aphidicolin. The aphidicolin blocked cells were chosenbecause the activity of the cyclin E-associated kinase is maximal at theG1 to S phase transition. Cell extracts were immunoprecipitated usingaffinity-purified anti-cyclin E antibodies and the immunoprecipitateswestern blotted using both CDC2 and CDK2 specific antisera. For allimmunoprecipitations the antibodies had been cross-linked to sepharose.Immunoprecipitations were carried out with pre-immune serum ("αPI"),blank sepharose beads ("SEPH"), affinity purified anti-p34 CDC2C-terminus ("αp34"), and affinity purified anti-cyclin E ("αE") (FIG.14A). The set of lanes labeled "-" contained no cell extract. Bothantisera were raised against peptides corresponding to the C-termini ofthe respective proteins. The C terminus of the CDC2 related proteins isnot highly conserved. The results show that the anti C-terminus CDC2antiserum recognized CDC2 and not CDK2, and conversely that theanti-C-terminus CDK2 antiserum recognized CDK2 and not CDC2 (FIG. 13).

Immunoblots of whole cell extracts show two forms of CDK2 (Rosenblatt etal., 1992; see also FIG. 14A). In both aphidicolin arrested cells (FIG.14A) and in exponentially growing cells (not shown; see FIG. 15) cyclinE preferentially associated with a more rapidly migrating form of CDK2.The identification of CDK2 in cyclin E immunoprecipitates has beenconfirmed using 3 different antisera independently raised against theC-terminus of human CDK2. All three antisera recognize CDK2 and not CDC2(Rosenblatt et al., 1992; Elledge et al., 1992; FIG. 13). The morerapidly migrating forms of CDK2 are currently believed to be more highlyphosphorylated (Rosenblatt et al., 1992). This is consistent with ourobservation that all the cyclin E-associated isoforms of CDK2 detectedin [³⁵ S]-methionine-labeled cell extracts were also detected inanti-cyclin E immunoprecipitates from [³² P]-orthophosphate-labeled cellextracts.

The results show that p34 CDC2 was also detected in the cyclin Eimmunoprecipitates although its abundance was substantially less thanthat of CDK2. In exponentially growing cells, a predominantlyhypophosphorylated form of p34 CDC2 was detected, while inaphidicolin-arrested cells there were also more highly phosphorylatedforms of p34 CDC2 associated with cyclin E (FIG. 14B). In both cases, itwas possible to detect only very small amounts of p34 CDC2 associatedwith cyclin E.

EXAMPLE 11 Cell Cycle Dependent Formation of a Cyclin E:CDK2 Complex

The phase in the cell cycle at which cyclin E and CDK2 form anenzymatically active complex was investigated. Exponentially growingMANCA cells were separated into 8 cell cycle fractions by centrifugalelutriation and cellular extracts prepared. The cell cycle position ofthe cells in each fraction was determined by flow cytometric measurementof nuclear DNA content (FIG. 15A). Cyclin E and its associated proteinswere immunoprecipitated using affinity-purified anti-cyclin Eantibodies. We visualized the presence of CDK2 by Western blotting usingan antiserum specific for the C-terminus of CDK2 (FIGS. 15B-15E). Theresults show that the level of enzymatically active cyclin E:CDK2complex peaked during late G1 and early S phase and declined inabundance as cells progressed through the remainder of the cell cycle.The abundance of the cyclin E:CDK2 complex closely corresponded to thecell cycle periodicity of the cyclin E-associated kinase (as describedin prior Examples). Furthermore, the present results suggest that inexponentially growing cells, cyclin E:CDK2 complexes did not accumulatein an inactive form prior to their activation in late G1. This patternof appearance and activation was observed to be similar to the patternreported for cyclin A-associated kinase activity, i.e., the activity ofwhich reportedly increased in direct proportion to the abundance ofcyclin A (Pines and Hunter, 1990; Marracino et al., 1992). However, thepresent results were different from those obtained with the cyclinB:p34CDC2 complex in that the cyclin B:CDC2 complex reportedlyaccumulates during S and G2, inactive and highly phosphorylated, priorto their activation at the onset of mitosis (Gould and Nurse, 1989;Pondaven et al., 1990; Solomon et al., 1990).

EXAMPLE 12 Abundance of Cyclin E is Cell Cycle Regulated

The abundance of the cyclin E protein was determined at different phasesof the cell cycle. MANCA cells were separated into fractionsrepresenting each stage of the cell cycle by centrifugal elutriation. Weanalyzed cell lysates from each fraction by immunoprecipitation usingaffinity-purified anti-cyclin E antibodies which we also used to measurethe abundance of cyclin E in the immunoprecipitates. The results showedthat cyclin E levels were maximal in late G1 and declined in S, G2 and M(FIGS. 15B-15E). The immunoassay procedure was found to accuratelyreflect the relative levels of cyclin E in each cell cycle fractionsince the amount of cyclin E protein detected was linearly dependent onthe amount of cell extract used in the immunoprecipitation (FIG. 15F).In sum, these results suggest that the abundance of the cyclin E:CDK2complex, and hence the periodicity of the cyclin E-associated kinaseactivity, may be directly regulated by the level of cyclin E.

EXAMPLE 13 Assembly of Cyclin E:CDC2 and Cyclin E:CDK2 Complexes invitro

As shown, cyclin E preferentially associates with p33 CDK2 rather thanp34 CDC2 in human cells. One possible explanation for this is that theaffinity of cyclin E is different for CDK2 than for CDC2. Thispossibility was evaluated in a cell-free system of recombinant cyclin Eand cell extracts containing CDC2 and CDK2 kinases. Cyclin E wasexpressed in Sf9 insect cells using baculovirus vectors. Cyclin Eprotein were over-expressed in the transducer insect cells, theintracellular concentrations was approximately 5-10 μM after 48 hours(Desai et al., 1992), and these cells were harvested and proteinsextracted for analysis. The binding between cyclin E, CDC2, and CDK2 wasevaluated using diluted insect cell extracts as a source for cyclin E,and extracts from G1 cells as a source of CDC2 and CDK2. To determinecell-cycle-dependent differences in the effects of cyclin E on the CDC2and CDK2 kinases, cell extracts were prepared from cells whose growthwas arrested for 12 hours (i.e., prior to S-phase) in media containing 2mM hydroxyurea (causing cells in S-phase to stop and all other cells topile up next to S-phase) followed by release of growth for 3.5 hours toallow all cells to enter S-phase ("HU" FIGS. 16A-16B); as well as, fromcells blocked with nocodazole, released for three hours into G1, andthen further selected by centrifugal elutriation ("G1", FIGS. 16A and16B). All cell extracts were prepared in hypotonic buffer. Theincubation mixtures were designed to bring the concentration of thethree proteins close to the normal physiologic levels at approximately0.2 μM. Diluted lysates containing the indicated cyclin E, CDC2, andCDK2 proteins were incubated alone or in combination for 30 minutes at37° C. under conditions suitable for in vitro replication of SV40 origincontaining plasmids (D'Urso et al., 1990). The formation of cyclinE:CDC2 and cyclin E:CDK2 complexes in the incubation mixtures wasdetermined using immunoprecipitation either with antisera to CDC2(anti-CDC2), the C-terminus of CDK2 (anti-CDK2), or cyclin E(anti-cyclin E) followed by SDS-PAGE, and autoradiography (FIGS. 16A,16B, 16C). The kinase activity associated with the different respectiveimmunoprecipitates was determined in the H1 kinase assay (as describedin the Examples, above).

The immunoprecipitates were tested for their ability to mediatephosphorylation of histone H1 (i.e., H1 kinase activity) by mixing theimmunoprecipitates with histone H1 and γ-³² P orthophosphate. ³²P-radiolabeled histone H1 was detected by SDS-PAGE and phosphor imaging(FIGS. 16A, 16B, 16C). (The phosphor imaging in FIGS. 16A-16C wasquantified and the results are graphically presented in FIG. 17.) CDC2kinase activity, while evident at low levels in immunoprecipitatesprepared from HU-arrested cells (FIG. 16C, "HU"), was decreased tonearly undetectable levels during G1-phase (FIG. 16C, G1 extract, "0")and the level of kinase activity was not altered by addition ofdifferent amounts of cyclin E to the cell extract (i.e., FIGS. 16A, 16B,16C; "5, 1, 0.2"). In contrast, CDK2 kinase activity present at lowlevels in HU-arrested cell extracts (FIG. 16A, "HU"), decreased toundetectable levels in G1 (FIG. 16A, G1 extract, "0"), but when cyclin Ewas added to the G1 cell extract (FIG. 16A, "5,1,0.2") the CDK2 kinaseactivity was restored. The results show activation of a latent CDK2kinase activity in the G1-phase cell extracts following addition ofcyclin E, and suggest that kinase activity is regulated by the abundanceof cyclin E. Quantitive aspects of these studies are presented in FIG.17, where the level of cyclin E-mediated activation of CDK2 kinaseactivity was measured (i.e., using phosphor imaging of the SDS-PAGE gelspresented in FIGS. 16A, 16B, and 16C, above) as a function of the amountof cyclin E added to the G1 phase cell extract ("fold cyclin E in HUextract"; FIG. 17). (The differing amounts of Sf9 lysate containingcyclin E in FIG. 17 correspond to the "5, 1, and 0.2" amounts in FIGS.16A, 16B, and 16C.) The phosphor imaging data for the kinase activity ofeach of the CDC2, CDK2, and cyclin E immunoprecipitates was normalizedby calculating the activity as a percentage of the activity seen in celllysates of HU-arrested control cells (i.e., 100%; "% hydroxyurea H1kinase"; FIG. 17). The results presented in FIG. 17 show that the levelof CDK2 kinase activity was dependent upon the amount of cyclin E addedto the G1 extract, and that levels of CDK2 kinase activity were achievedwhich were more than 22-fold greater than those observed in theHU-arrested cell extracts (i.e., cyclin E immunoprecipitate at 5-foldcyclin E; FIG. 17). In addition, the results show that the kinaseactivity associated with the cyclin E-immunoprecipitates wasconsistently greater than that associated with CDC2 immunoprecipitates.The results also confirm the previous findings (above) that only lowlevels of CDC2 activity are present in the G1-phase cell extracts, andthat any latent CDC2 that might be present in these extracts is notappreciably activated by the addition of cyclin E.

These combined results suggest activation of kinase activity by cyclin Eresulting from formation of a cyclin E:CDK2 complex. In other studies(not shown) the association of cyclin E with CDC2 or CDK2 was verifiedusing molecular-sieve gel chromatography on Superose 12. p34 CDC2 andp33 CDK2 monomers eluted at 30-40 kDa and had negligible histone H1kinase activity. When insect cell extracts containing recombinant cyclinE were mixed with the CDK2-containing lysate, the majority of the CDK2protein eluted at an approximate molecular size of 160 kDa, suggestingformation of a cyclin E:CDK2 complex. In contrast, when extractscontaining a similar amount of CDC2 were mixed with the cyclin E lysateonly a small fraction of CDC2 protein associated in a stable manner withcyclin E. The cyclin E:CDC2 and cyclin E:CDK2 complexes eluted from themolecular sieve column exhibited kinase activity.

DISCUSSION OF EXAMPLES 8-13

Cyclin E is a G1 Cyclin.

The proliferation of eukaryotic cells is primarily regulated by a singledecision which occurs during the G1 phase of the cell cycle--either toenter the cell cycle and divide or to withdraw from the cell cycle andenter a quiescent state (reviewed in Baserga, 1985; Pardee, 1989). Inyeast, the biochemical process that underlies this cellular decision isthe assembly and activation of a complex between the CDC8 protein kinaseand the CLN type cyclins (reviewed in Nurse, 1990; Hartwell, 1992).Recent experiments in a variety of model systems support the idea thatthe role of the CDC2-related kinases have been evolutionarily conserved(D'Urso et al., 1990; Blow & Nurse, 1990; Furakawa et al., 1990; Fang &Newport, 1991). The observations presented here demonstrate that humancyclin E specifically activates a CDC2 related kinase during the late G1phase of the cell cycle and that cyclin E accumulation is rate-limitingfor G1 transit. Therefore, we suggest that in all eukaryotes a criticalstep in the biochemical pathway that controls cell proliferation is theassembly of a cyclin/CDK complex (the term CDK is used to designate acyclin dependent kinase in the CDC2 protein family).

The evidence that cyclin E functions during the G1 phase of the humancell cycle can be summarized as follows: Cyclin E can perform the G1START functions of the yeast CLN proteins since it can complementmutations in the yeast CLN genes (Koff et al., 1991; Lew et al., 1991 ).Furthermore, we have shown that cyclin E in combination with eitherhuman CDC2 or human CDK2 could rescue yeast strains that were doublymutated for both CLN and CDC28 function (Koff et al., 1991). However,the specificity of the assay was suspect since human cyclin B, whichclearly functions during mitosis and not during G1, could also rescueCLN mutations (Koff et al., 1991; Lew et al., 1991; Xiong et al., 1991). As reported here, cyclin E associates with a protein kinase in humancells and this kinase is cell cycle regulated. The activity of thecyclin E-associated protein kinase, as well as the abundance of thecyclin E protein, peaks during late G1 and early S phase, and thendeclines as cells progress through S, G2 and mitosis. This kinase isalso growth regulated, since it is absent from cells that have exitedthe cell cycle and differentiated or become quiescent. The relativetiming of cyclin E and cyclin A activity is significant. Cyclin Aprotein and cyclin A-associated kinase activity are detectable as soonas S phase starts (Marraccino et al., 1992), and cyclin A function isnecessary for S phase to begin (Girard et al., 1991). We have also shownthat cyclin E accumulates before cyclin A and that the cyclinE-associated kinase appears earlier in the cell cycle than the kinaseassociated with cyclin A. This biochemical function of cyclin E duringG1 suggested that its physiological function would precede the S phaserole of cyclin A. This was directly shown by constitutively expressinghuman cyclin E in the rat fibroblast cell line, Rat-1. We found that 5to 10-fold overexpression of cyclin E caused a 3 to 5-fold increase inthe level of cyclin E-associated kinase activity. This level of cyclin Eoverexpression caused a 30-35% decrease in the length of the G1 phase ofthe cell cycle.

The abundance of the cyclin E protein is normally cell cycleregulated--it shows a sharp peak in late G1. This is probably due toregulation of the cyclin E mRNA level (Lew et-al., 1991) since itfluctuates during the cell cycle in parallel with the level of thecyclin E protein. The mRNA's encoding cyclin E, A and B are cell cycleregulated and predict the pattern of accumulation of the respectivecyclin proteins (Pines and Hunter, 1989, 1990). In budding yeast,accumulation of the CLN mRNA's is under positive feedback control andresult in a rapid rise in CLN mRNA and protein levels at START (Crossand Tinkelenberg, 1991; Dirick & Nasmyth, 1991). The association ofcyclin proteins with transcription factors in mammalian cells may bepart of an analogous mechanism that controls the timing of cyclin geneexpression during the cell cycle (Bandara et al., 1991; Mudryj et at.,1991; DeVoto et al., 1992; Shirodkar et al., 1992). While cyclinaccumulation is in part determined by the levels of the respectivemRNA's, cyclin abundance can also be controlled by protein turnover(Murray & Kirschner, 1989; Glotzer et al., 1991). It is not knownwhether the stability of the cyclin E protein is regulated during thecell cycle, but the protein lacks the consensus sequence recognized bythe ubiquitinating enzyme that mediates the mitotic turnover of cyclinsA and B (Glotzer et al., 1991).

The Cyclin E:CDK2 complex

The data suggest that the major cyclin E-associated protein kinase isCDK2. Two dimensional gel analyses of ³² P or ³⁵ S-met labelled proteinsshow that the major CDC2-related protein associated with cyclin E inhuman cells is CDK2. While there is not direct evidence that the cyclinE:CDK2 complex is an active kinase in vivo, this is the most likelyconclusion. The abundance of the cyclin E:CDK2 complex is cell cycleregulated and closely parallels the levels of the cyclin E-associatedkinase. Furthermore, the CDK2 protein bound to cyclin E is primarily inthe more rapidly migrating of the two forms detectable by onedimensional PAGE. This downward mobility shift is known to correlatewith both binding of CDK2 to cyclin and activation of the CDK2 kinase(Rosenblatt et al., 1992). It is thought to be indicative ofphosphorylation of threonine 160, which is a prerequisite for activationof the CDK2 kinase (Y. Gu and D. M., unpublished observations). It hasalso been shown that the cyclin E:CDK2 complex can substitute for theCLN/CDC28 complex in S. cerevisae (Koff et al., 1991), and that thecyclin E:CDK2 complex is an active kinase in vitro.

The periodic accumulation of the cyclin E protein in cells appears tomatch that of the cyclin E:CDK2 complex, whereas the CDK2 protein ispresent at invariant levels during the cell cycle (Rosenblatt et al.,1992). Therefore, it would seem that the abundance of the cyclin E:CDK2complex is primarily regulated by the level of the cyclin E protein.However, the phosphorylation state of cyclin E could also control theassembly of the complex.

At least six phosphorylated isoforms of CDK2 are associated with cyclinE. This complexity was surprising since only two of these isoforms hadbeen detected bound to cyclin A. Preliminary evidence indicates thatCDK2 is phosphorylated on 3 residues homologous to those phosphorylatedin CDC2 (Y. Gu and D. M., unpublished observations)--T14, Y15 and T160.Combinatorial phosphorylation of these sites might account for the sixCDK2 isoforms. It seems more likely, however, that other phosphorylationsites are also present since immunoprecipitates with anti-CDK2antibodies contained two additional phosphorylated isoforms of CDK2,bringing the total number detected to eight (FIG. 11A). Oneinterpretation is that the cyclin E:CDK2 complex integrates theinformation provided by the many signals that control cellproliferation, e.g., by binding second messengers involved in signaltransduction. The multiple phosphorylated forms of CDK2 may reflect theinfluence of diverse mitogenic signals on the activation of the cyclinE:CDK2 complex. The multiple CDK2 phosphates could have both positiveand negative effects on CDK2 activity and a particular phosphorylatedstate may be required for specific functions. The downstream activationof the cyclin A:CDK2 complex, which occurs after commitment to the cellcycle has been made, may be responsive to much fewer factors andtherefore biochemically less elaborate.

Other evidence has indicated that CDK2 might play a role during the G1of S phases of the cell cycle. In cycling cells, CDK2 kinase activityprecedes CDC2 kinase activity (Rosenblatt et al., 1992). Our experimentsin S. cerevisae showed that in certain genetic backgrounds CDK2 cancomplement the G1/S function of CDC28 and not its G2/M functions (Koffet al., 1991). Also, depletion of CDK2 from extracts of activatedXenopus eggs prevents the start of DNA replication (Fang and Newport,1991). All these results are consistent with a role for CDK2 incommitting the cell to the cell cycle.

The Cyclin E:CDC2 Complex.

Cyclin E can interact with both human CDK2 and human CDC2 when theproteins are expressed together in yeast and cyclin E can activate boththe CDK2 and CDC2 kinases in vitro (Examples, above). We have shown thatalthough the cyclin E:CDK2 complex is more abundant in human cells, thecyclin E:CDC2 complex is also present. In addition, complexes betweencyclin E and other proteins were observed (FIG. 12; Table 1) that maypotentially modulate or change cyclin E activity. The pattern of cyclinE-associated kinase activity during the cell cycle showed somedifferences from the abundance of the cyclin E:CDK2 complex. Thesedifferences may be attributable to the cyclin E:CDC2 or the other cyclinE complexes.

The low level of the cyclin E:CDC2 complex in vivo appears to be aconsequence of the relatively low affinity of cyclin E for CDC2. Thereconstitution experiments presented here show that the cyclin E:CDK2complex readily formed under conditions where very little cyclin E boundto CDC2. We have found, however, that cyclin E is present in multiplephosphorylated states in vivo. Therefore, another possibility is thatonly certain relatively rare isoforms of cyclin E can bind to CDC2.

We previously observed that a mutation in the yeast CDC28 gene greatlycurtailed the ability of cyclin E, but not cyclin B, to rescue CLNfunction and, consequently, we suggested that cyclin E might interactwith CDC28 differently than cyclin B (Koff et al., 1991). In vitroreconstitution experiments support this idea by showing that cyclin Bbound to CDC2 effectively (Desai et al., 1992) while only small amountsof cyclin E:CDC2 complex could be detected.

Other Cyclin E:CDC Complexes

The results presented in FIG. 12 and Table 1, above, may also beinterpreted to indicate the possible existence of other cell divisionkinases, previously unrecognized, that associate with cyclin E. The 32Kd band "x" protein (Table 1, FIG. 12) is certainly a candidate for sucha novel kinase protein, both based on the similarity in size with theknown CDC2 and CDK2 kinases, and its apparent association with cyclin E.

G1 Regulation in Mammalian Cells

In 1974 Pardee proposed that the proliferation of mammalian cells isregulated by extracellular mitogenic signals at a point during the G1phase of the cell cycle called the restriction point (Pardee, 1974). Ifthese signals were not present, or if the cell was incapable ofappropriately responding to them (e.g., if protein synthesis isinhibited) then the cell would not traverse the restriction point andentered a quiescent state, called G₀ (reviewed in Zetterberg, 1990).While cells can respond to a wide array of extracellular mitogenicsignals, one gets the impression that there is much less diversity inthe intracellular events triggered by these signals (see Cantley, 1991;Chao, 1992). Indeed, it is not unreasonable to expect that there mightbe a final common point through which the diverse mitogenic pathwaysmust pass, and that this is the restriction point (Pardee, 1974).

There are few molecular details about restriction point regulation. Innormal cells, progression through the restriction point is verysensitive to the rate of protein synthesis (Rossow et al., 1979,Schneiderman et al., 1971; Brooks, 1977). Prior to the restriction point(but not after) small and transient decreases in protein synthesis causesubstantially longer increases in the length of G1 (Zetterberg & Larson,1985). Removal of extracellular mitogenic stimuli and inhibition ofcellular protein synthesis, in fact, are thought to deter the same cellcycle event (Pardee et al., 1981). To account for the disproportionatelylarge effect on G1 length by relatively small changes in the rate ofprotein synthesis, it was proposed that a labile protein must accumulateduring G1 in order for the cell to traverse the restriction point(reviewed in Pardee, 1989).

It is appealing to speculate that a cyclin is this labile regulator ofthe restriction point and that formation and/or activation of acyclin/CDK complex is a rate-limiting event in G1 progression. Theperiodic accumulation of cyclin E during the cell cycle indicates thatit is a relatively short lived protein, and its G1 peak in abundance maybe consistent with a role at the restriction point. Also, the decreasein G1 length by constitutive cyclin E expression suggests that entryinto S phase may be limited by the abundance of cyclin E. It isimportant to remember, however, that not all of G1 is eliminated byconstitutive cyclin E expression. Most likely, there are some essentialG1 events whose duration is not effected by abundance of cyclin E.Examples of this might include chromosome decondensation and nuclearmembrane assembly. Furthermore, it has been reported that in somecircumstances the G1 restriction point occurs less than one hour beforeS phase starts (Wynford-Thomas et al., 1985) while in other cases therestriction point can occur much earlier in G1 (Pardee, 1974). Ourmeasurements indicate that the maximal cyclin E-associated kinase levelsare reached relatively late in G1. This is particularly apparent inserum stimulated cells where much of G1 is completed before the cyclinE-associated kinase is detected (A. K. and J. R., unpublishedobservations). Other cyclins, such as cyclin D and cyclin C, may also beexpressed during G1 (Matsushime et al., 1991; Motokura et al., 1991; Lewet al.; 1991) and it is possible that the sequential formation ofmultiple cyclin:CDK complexes is required for the cell to traverse G1.In that case, constitutive cyclin E expression might shorten only thelatter stages of G1.

In S. cerevisae factors that control passage through START can effectCLN function, apparently at multiple levels (Change & Herskowitz, 1990;Cross and Tinkelenberg, 1991). By analogy, we might expect G1 cyclinfunction in mammalian cells to be controlled by proteins that modulatecell proliferation. For example, it would not be surprising to observedirect interactions between cyclin E and members of the Rb proteinfamily (Bandara et al., 1991; Mudryj et al., 1991; Shirodkar et al.,1992; DeVoto et al., 1992). Also, expression of the cyclin E gene mightbe regulated by one or more of the oncogenic transcription factors.

EXAMPLE 14 Growth Factor-dependence of Cells Constitutively ExpressingCyclin E

The cell division cycle of all normal higher eukaryotic cells iscontrolled by specific extracellular growth factors that are requiredfor cell division. The families of known growth factors is diverse andincludes such proteins as insulin, PDGF, IGF, EGF, GM-CSF, G-CSF, TGF,erythropoeitin, and other stem cell factors. Different cell typesdisplay particular growth factor requirements, determined in part by thegrowth factor receptors expressed on their cell surface and by theirstate of differentiation. Typically, cells in tissue culture requireexogenous growth factors in an animal serum (i.e., fetal bovine serum)to grow; or in chemically defined serum-free medium specific growthfactors must be added. In the absence of the requisite growth factor(s),cells stop dividing and arrest in G1. The results presented in theExamples above indicated that the level of cyclin E, and/or the activityof the cyclin E:cell division kinase complex, may be rate-limiting fortransit of cells through G1. Therefore, it was reasoned that cellproliferation might be regulated through steps requiring cyclinactivation (e.g., increased cyclin gene transcription or translation; orincreased cyclin:kinase complex activity) and that growth factors mightact upon cells by activating cyclins. Assuming that cyclin activation isrequired for proliferation two hypotheses were considered: a unitaryhypothesis in which a cell at a particular stage of differentiation hasa single cyclin that can be triggered by a single growth factor; and amultiform hypothesis, wherein a single growth factor activates multiplecyclins and the combined action of all the cyclins in the cell isrequired to trigger cell proliferation. It was reasoned that if thesimple cause-and-effect logic of the unitary hypothesis held true, thenmodifying cyclin E levels in a cell might alter the growth factorrequirements of the cell for proliferation in vitro; while if themultiform hypothesis held true, then any alteration in a single cyclinmight be masked by the action of all the other cyclins in the cell. Totest these two hypotheses, cells were transducer with the LXSN-cyclin Evector sequences.

Primary cultures of human fibroblasts and rat Rat-1 cells were infectedwith LXSN-cyclin E vector particles, or as a control with LXSN (asdescribed in Example 9). The transduced cells were tested for expressionof cyclin E (as described above), and LXSN-cyclin E-transduced Rat-1 andhuman fibroblasts were found to express 3- to 5-fold greater levels ofcyclin E protein than the cells from which they were derived (and 3- to5-fold greater than control LXSN-transduced cells). The growth-factordependence of LXSN-cyclin E-transduced human cells was determined bymeasuring tritiated thymidine incorporation into DNA in serum freemedium (D-MEM) or medium supplemented with 10%, 1%, 0.1%, or 0.01% (v/v)fetal bovine serum (Table 2) and the growth factor dependence of Rat-1cells was determined by measuring BrdU incorporation in 10%, 1.0% or0.1% serum (FIGS. 18A-18B). In the BrdU assay, only cells that aresynthesizing DNA (i.e., S-phase cells) incorporate BrdU into DNA andscore positive in the assay. Therefore, the rate of accumulation of BrdUpositive cells can be taken as a relative measure of the rate at whichcells transition from the conclusion of one mitosis through G1-phase andinto the next round of DNA synthesis (i.e., S-phase). The resultspresented in FIGS. 18A and 18B show the percent of the total cell nucleithat were labeled with BrdU in cultures of LXSN-transduced control Rat-1cells ("RAT1/LX", open circles) and LXSN-cyclin E-transduced cells("RAT1/cyclin E", closed circles) as a function of time after releasingmitotic arrest induced by nocodazole treatment. The growth factordependence of the cells was evaluated by culturing the cells in 10%bovine calf serum (FIG. 18A) or in 1% or 0.1% serum (FIG. 18B). Theresults in FIGS. 18A and 18B show that a) irrespective of the percentageof serum in the culture medium, the LXSN-cyclin E-transduced cellsinitiated DNA synthesis more rapidly than control cells: and, b) theLXSN-cyclin E-transduced cells exhibited increased resistance to lowserum (i.e., 0.1%) and initiated DNA synthesis about 10-12 hours soonerthan the control cells (FIG. 18B).

In a similar manner, the results presented in Table 2 show thatLXSN-cyclin E-transduced human fibroblasts and Rat-1 cells exhibitreduced growth factor requirements for proliferation. In control cells(i.e., LXSN-transduced cells) when serum was reduced from 10% to 0.1%the cells continued to proliferate and incorporate thymidine into DNA,although at a reduced rate, with levels 11% of those observed in thepresence of optimal levels of growth factors (i.e., 10% serum). Incontrast, the growth of LXSN-cyclin E-transduced cells was reduced to19% of maximal levels (seen in 10% serum) but this level was more than2-fold higher than the level observed in the control LXSN-transducedcells. In addition, when PDGF (10 ng/ml) was added to cyclinE-transduced cells growing in 0.1% serum, the levels of proliferationwere 50% of the maximal level occuring in the presence of 10% serum(Table 2). In contrast, when PDGF was added to control human fibroblastsgrowing in 0.1% serum no stimulation of thymidine incorporation wasobserved.

                  TABLE 2                                                         ______________________________________                                        Growth Factor Dependence of Vector-Transduced                                 Human Fibroblasts                                                             Transducing                                                                              Serum             .sup.3 H-TdR                                                                         Percent                                   Vector     (%)     PDGF.sup.a                                                                              (CPM).sup.b                                                                          Max CPM.sup.C                             ______________________________________                                        LXSN       10      0         524,300                                                                              100                                                  0.1     0         59,359 11                                                   0.1     +         55,788 11                                        LXSN-cyclin E                                                                            10      0         708,871                                                                              100                                                  0.1     0         137,712                                                                              19                                                   0.1     +         356,284                                                                              50                                        ______________________________________                                         a.) + = PDGF (10 ng/ml) added to the culture media; 0 = no PDGF;              b.) .sup.3 HTdR, tritited thymidine incorporation determined 36 hours         after adding 1-2 μCi/ml .sup.3 HTdR to culture medium;                     c.) Percent Max CPM, % maximal .sup.3 HTdR CPM = (CPM in 0.1%)/(CPM in 10     serum)                                                                   

(Flow cytometric analysis confirmed the continued presence of cyclingcells in LXSN-cyclin E-transduced Rat-1 and human fibroblast cells inthe presence of 0.1% serum.) The combined results show that theLXSN-cyclin E-transduced Rat-1 cells have a reduced growth factordependence for transitioning the G1 phase of the cell cycle.

These combined results support the hypothesis that over-expression by3-5 fold of a single cyclin, i.e., cyclin E, can partially (but notcompletely) restore the ability of cells to proliferate in the absenceof growth factors, and fully restore proliferation in the presence of asingle growth factor, PDGF. Thus, the results tend to favor a unitaryhypothesis in which one cyclin and one growth factor regulate growth ofa cell at a particular stage of differentiation; however, thisinterpretation is not supported by the quantitative aspects of the data,i.e., over-expression did not render the cells completely growth factorindependent. Therefore, the possibility also exists that cyclins otherthan cyclin E are participating in the stimulation of cell proliferationin the presence of PDGF. (The results could thus be interpreted asproviding support for a multiform model of cell proliferation whereactivity of several cyclins and growth factors combines to promote cellproliferation.)

In summary, the results can be interpreted to provide support for eithera unitary or multiform model of cell proliferation. Aside from anyinterpretations, the results are significant for demonstrating thatgenetic manipulation of a single cyclin in a cell and treatment with asingle growth factor is sufficient to dramatically alter the conditionsrequired to grow cell in vitro. It appears from the results that withthe proper combination of G1 cyclin expression in a particular cell: a)a cell line may be produced whose proliferation is largely unconstrainedin the absence of exogenous growth factors; and b) a cell line may beproduced whose proliferation is largely dependent upon one or moreselected growth factors. In viewing the potential long-term significanceof the present findings it may be worthwhile to recall that it took SamHanks nearly 10 years of research to develop Hank's Balanced SaltSolution (HBSS); about an additional 3-5 years for Eagle to developEagle's Minimal Essential Medium (MEM); and still longer for ReneDulbecco to achieve a D-MEM formulation. (Media such as RPMI 1640 andM199 still carry a number that designates how many formulations precededtheir development.) The findings described herein are thus highlysignificant, for showing that simple manipulation of a single protein ina cell is sufficient to promote propagation of the cell in vitro in thenear complete absence of serum growth factors.

MATERIALS AND METHODS

Plasmids and libraries 1858

The human cDNA library was a gift from J. Colicelli and M. Wigler. Itwas prepared from the human glioblastoma cell line U118 in the vectorpADNS (Colicelli et al., 1988). The portion of the library used in theseexperiments contained cDNA inserts that had been selected to be >2 kb.In the experiments involving isolation of human CDC2 homologs, thecyclin E cDNA was transferred to the vector pMAC. This 2μ-based vectoruses the ADH promoter to drive expression of the human cDNA and containsthe TRP1 selectable marker. For expression of cyclin E in E. coli aSmaI-PvuII fragment containing the entire cyclin E coding region wascloned into the unique SmaI site in the vector pGEX-3T (Amgen). For invitro transcription/translation reactions, the SmaI-NotI fragment ofcyclin E was cloned into the MscI site in the vector pCITE-1 (Novagen).For in vitro translation of human cyclins A and B, cDNAs withgenetically engineered NcoI sites at the initiating methionine weregenerously provided by Jonathan Pines and Tony Hunter. PCITE vector wascleaved with Sal I, blunt-ended with Klenow enzyme and then cleaved atthe unique NcoI site. Cyclin cDNAs were isolated by cleavage with EcoRI(for cyclin A) or BamHI (for cyclin B), blunt-ended with Klenow and thencleaved with NcoI. The Xenopus CDK2 clone, pEMBLYe30/2, has beendescribed previously (Paris et al., 1991). For some assays in yeast, thecyclin A, B, and E cDNAs were subcloned into the vector pADANS, which isidentical to pADNS except that the first 10 amino acids of the ADHprotein are fused to the expressed protein.

Antibodies

The peptide YLDNQIKKM (SEQ. ID. NO. 3), corresponding to the C terminusof human CDC2, was synthesized chemically and covalently coupled to BSAvia the tyrosine residue for injection into rabbits. For affinitypurification, rabbit serum was precipitated with 50% ammonium sulfate,resuspended in 10 mM sodium phosphate (pH 8.0) and dialyzed extensivelywith 10 mM sodium phosphate, 0.15M NaCl, pH7.2 (PBS). Affinity columnswere prepared by coupling the peptide to CNBr-activated Sepharose usingconditions recommended by Pharmacia. The dialysate was applied to theaffinity column equilibrated in PBS. The follow-through was subsequentlyreloaded twice. The column was washed with 10 column volumes of PBS+2MKCl, and protein subsequently was eluted with 10 column volumes of 5MNaI+1 mM sodium thiosulfate (made fresh before use). Fractionscontaining immunoglobulin were determined by absorbence at 290, pooled,and dialyzed extensively against PBS. The peptide CEGVPSTAIREISLLKE(SEQ. ID. NO. 4), corresponding to the conserved "PSTAIRE" domain of theCDC2 gene family, was synthesized chemically and coupled to keyholelimpet hemocyanin (KLH) via the cysteine residue, and antibodies wereprepared in rabbits and affinity purified as described above. Thepeptide YDEAEKEAQKKPAESQKIERE (SEQ. ID. NO. 5), corresponding toresidues 104-123 of human cyclin A, was synthesized chemically andcoupled to BSA, and antibodies were prepared in rabbits and affinitypurified as described above.

Antibodies directed against CDK2 were raised against a peptidecorresponding to the 15 C-terminal amino acids of human CDK2 coupled tokeyhole limpet hemocyanin. Two other antisera against the 9-C-terminalamino acids of human CDK2 were also used in the course of theseexperiments (Elledge et al., 1992; Rosenblatt et al., 1992). Thepolyclonal anti-cyclin E antisera has been described (Koff et al.,1991).

For preparation of cyclin E antibodies, E. coli containing GEX-cycE (seebelow) were grown to an OD₆₀₀ of 0.4-0.6, and fusion protein expressionwas induced with 10 mM IPTG. After 3 hours of additional growth at 30°C., the E. coli were pelleted, washed once with PBS, and again with GEXbuffer A (60 mM Tris-HCl pH 8.0, 25% sucrose, 10 mM EDTA) and stored at-75° C. Cells were resuspended in 1/30 the original culture volume inGEX buffer A containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 10μg/ml leupeptin, 100 μg/ml soybean trypsin inhibitor (SBTI), and 10μg/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). Proteaseinhibitors were used in all subsequent steps. SDS was added to 0.03% andcells were lysed by sonication. Lysates were clarified by centrifugationat 13,000×g and added to a 1:1 slurry of Sepharose CL4B in GEX buffer C(0.02M HEPES-KOH (pH 7.6), 100 mM KCl, 1.2 mM EDTA, 20% glycerol, and 1mM DTT) with 0.03% SDS, incubated for one hour at 4° C., and theSepharose removed by low speed centrifugation. Cleared lysates wereincubated with glutathione-agarose beads (SIGMA #G4510) (approximately360 μg of GEX-cyclin E per ml of glutathione-agarose beads) for 1 hourat 4° C. The agarose beads were pelleted and washed 5 times with 10volumes of GEX buffer C with 0.03% SDS, and the cyclin E fusion protein(GEX-E) eluted with buffer C with 0.03% SDS plus 5 mM glutathione.Fractions containing GEX-E were identified by SDS-PAGE electrophoresisand Coomassie blue staining. Rabbits were injected with 400 μg of totalGEX-E protein in complete Freund's adjuvant; 320 μg was injectedsubcutaneously and 80 μg intramuscularly. Rabbits were boosted every 3weeks with an identical regimen except incomplete Freund's adjuvant wasused. Bleeds were obtained 7 days postinjection and analyzed by theirability to immunoprecipitate cyclin E produced in a rabbit reticulocytelysate (Promega).

The specificity of the cyclin E antiserum was demonstrated byimmunoprecipitation of in vitro translated cyclin E, A, and B. In vitrotranslated cyclins were made according to manufacturer's directions.Briefly, plasmids were linearized with either NheI (cyclin B/cyclin E)or PstI (cyclin A). Cyclin A was subsequently blunt ended with theKlenow enzyme before the transcription reaction. Transcription wascarried out using the T7 RNA polymerase, and RNA was isolated by ethanolprecipitation. Rabbit reticulocyte lysates were programmed with the RNAand incubated for 2 hours at 30° C. Programmed lysate (5 μl) wasincubated with 10 μl of cyclin E antisera in 500 μl of 50 mM Tris-HCl pH7.4, 250 mM NaCl, and 0.1% NP-40 for 1 hour at 4° C. Protein A-Sepharosewas added and incubation continued for 1 hour. Protein A beads werepelleted and washed 4 times with 50 mM Tris-HCl, pH7.4, 10 mM MgCl₂, 1mM DTT, and 0.1 mg/ml BSA. The immunoprecipitates were resuspended insample buffer and run on 12% SDS-PAGE gels. The gels were fixed andenhanced with 1M sodium salicylate before drying and autoradiography.

Cyclin E antibodies were affinity purified on columns of GST-cyclin Efusion protein. Approximately 100 ml of rabbit sera was precipitatedwith 50% ammonium sulfate. The precipitate was collected at 8,000×g andresuspended in 10 mM sodium phosphate pH 8.0 and dialyzed against PBS.The dialysate was adjusted to 10% glycerol and pre-cleared over aglutathione-S-transferase (GST) column. Flow through fractions werecollected and the column regenerated by washing with 0.2M glycine pH2.2. The column was re-equilibrated with PBS and this process wasrepeated 3 times.

Cleared sera was subsequently applied to a GST-cyclin E column.Following adsorption, the column was washed first with PBS and then with2M KCl-PBS, and bound antibody was eluted with NaI-sodium thiosulfate asdescribed (Koff et al., 1991). The eluate was dialyzed against couplingbuffer (0.1M NaHCO₃ pH 8.3, 0.5M NaCl) and concentrated 5 to 10-foldusing Centricon 10 concentrators (Amicon).

DNA Sequencing

Nested deletions of the cyclin E cDNA were sequenced on both strandsusing dideoxy chain termination methods.

Kinase assays

GEX-cyclin E (GEX-E) was purified as described up to the washing of theGEX-E bound to glutathione-agarose. For this experiment the beads werewashed 3 times with 5 volumes of GEX buffer C with 0.03% SDS, 5 timeswith 10 volumes of buffer C with 0.5% Triton X-100, 5 times with 10volumes of buffer D (30 mM HEPES-KOH pH 7.6, 7 mM MgCl₂, 100 mM NaCl, 1mM DTT). 100 μl of GT-cyclin E-Sepharose beads, p13-Sepharose (5 mg ofp13 per ml Sepharose), GT-Sepharose, or blank Sepharose were incubatedwith 100 μg of S-100 extract from human MANCA G1 cells (Roberts &D'Urso, 1988) in conditions used for in vitro replication of SV40 DNA(buffer D plus 3 μg creatine phosphokinase, 40 mM phosphocreatine, 0.25mM dNTPs, 0.5 mM CTP, UTP, and GTP, 3 mM ATP). The beads were thenpelleted and washed 5 times in kinase buffer (50 mM Tris-HCl pH 7.4, 10mM MgCl₂, 1 mM DTT) plus 0.1 mg/ml BSA. For kinase assays the beads wereresuspended in 50 μl kinase buffer+30 μM ATP, 5 μCi γ-³² P-ATP, and 1 μghistone H1, and incubated at 37° C. for 30 minutes. Products wereanalyzed by SDS-PAGE followed by autoradiography.

For studying the kinase bound to the SDS-GT-cyclin E-Sepharose beads,the GT-cyclin E beads and GT beads were prepared and incubated with G1extracts and washed as described. Incubation of the beads at 37° C. for30 minutes in TNT (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20)and 5 mM glutathione (reduced form) was sufficient to release theproteins bound to the beads by interaction with glutathione. Thesupernatant was transferred to a fresh tube and immunoprecipitated withaffinity-purified antisera directed against the C-terminus of p34 cdc2.The immunoprecipitates (with Protein A-Sepharose) were washed threetimes in TNT and used in a histone H1 kinase assay as described.

To show phosphorylation of the GT-cyclin E protein by the bound CDC2kinase, GT-cyclin E beads were prepared and incubated with G1 extractsand used in a kinase assay as described previously for histone H1;however histone H1 was not included in the assay. After incubation thepellet is washed with H1 kinase buffer 0.1 mg/ml BSA, then with 30 mMHEPES-KOH pH 7.5, 7 mM MgCl₂,1 mM DTT, 0.1 mg/ml BSA 0.2M NaCl, andfinally with TNT. The beads were then incubated with 1 ml of TNT and 5mM glutathione (pH 7.5) at 37° C. for 30 minutes to release theGT-cyclin E fusion protein. The supernatant was then collected andimmunoprecipitated with antisera directed against cyclin E. Immunecomplexes were subsequently collected by adherence to ProteinA-Sepharose. Immunoprecipitates were washed 3 times with TNT andproducts analyzed on 12% SDS-PAGE gels followed by autoradiography.

For immunoprecipitation of cyclin E from HeLa cell extracts, 2×10⁶ cellswere lysed in 50 mM Tris-HCl pH 7.4, 250 mM NaCl, and 0.1% NP-40 andclarified by ultracentrifugation at 100,000×g for 30 minutes. Sampleswere immunoprecipitated using Protein A-sepharose with 15 μl of normalrabbit sera or sera generated against the cyclin E fusion protein.Immunoprecipitates were washed with kinase buffer and 0.1 mg/ml BSA, andthe kinase assay was performed as described above.

T-peptide kinase assays were performed as described previously (D'Ursoet al., 1990).

Yeast strains:

Yeast strains used were isogenic with strain YH110 (Richardson et al.,1989). Unmarked deletions of CLN2, CLN2, and CLN3 were constructed inthis strain background. These deletions removed significant portions ofthe cyclin homology in CLN1 and CLN2 (Hadwiger et al., 1989; Cross &Tinklenberg, 1991) and completely deleted the CLN3 coding sequence(Cross, 1990). All the deletion alleles were null alleles by the assaysdescribed previously (Richardson et al., 1989). These deletion alleleswere unmarked, unlike the originally described cln disruptions(Richardson et al., 1989), and therefore were compatible with theplasmid transformation experiments performed here. The cln-deficientstrain was kept alive by the GAL-CLN3 plasmid described previously(Cross, 1990). The cdc28-13 allele in this isogenic strain backgroundwas provided by D. Lew and was combined with the three cln deletions bymating and tetrad analysis.

Yeast transfections

Transfections were performed using the lithium acetate procedureaccording to the method of Schiestl and Gietz (1989). Yeast cells grownin galactose were transfected with 2 μg of library DNA in each of 50independent aliquots. Transformants were selected on galactose forleucine prototropy and typically numbered 1000-2000 per plate. Colonieswere grown for 2 days and then replica plated onto YEP-glucose. Coloniesthat grew on glucose were patched onto FOA medium (Boeke et al., 1984)to identify colonies that could grow without the GAL-CLN3 plasmid.Plasmid DNA was rescued into E. coli by electroporation from coloniessurviving this screen and minipreps were retransfected into 589-5 strainyeast cells to confirm plasmid-dependent complementation of the triplecln deletion. For the screen identifying human CDC2 homologs, coloniesgrowing on glucose were tested for cosegregation of glucose growth andretention of the transfected plasmids.

Construction of cyclin E retrovital vector

The cyclin E retroviral vector (LXSN-cyclin E) was constructed byinserting a blunt-ended HindIII fragment of the human cyclin E cDNA HU4(Koff et al., 1991) (which contains the entire open reading frame) intothe HpaI site of LXSN, a murine retrovirus-based vector (Mill andRosman, 1989), in the sense orientation.

Cells

MANCA cells were maintained at 2-5×10⁵ cells/ml in RPMI plus 10% calfserum in an atmosphere containing 5% CO₂. Cells were fractionated fromexponentially growing populations by centrifugal elutriation (Marraccinoet al., 1992). For synchronization at the G1/S boundary approximately1×10⁸ G1 cells were collected from exponentially growing populations ofMANCA cells by elutriation and inoculated into RPMI containing 10% calfserum and 5 μg/ml aphidicolin and allowed to grow for 8 hours. Flowcytometric measurement of cellular DNA content was used to demonstratethe synchrony of the cell population. MANCA cells synchronized in G1were prepared exactly as described previously (Marraccino et al., 1992).

Rat PC-12 cells were maintained in DMEM containing 5% fetal calf serumand 10% horse serum in an atmosphere containing 10% CO₂. To induceneuronal differentiation confluent cells were split 1:20 and on thesecond day the media was replaced with serum free medium. Cells wereincubated in serum free media for 24 h and the medium was then changedto complete medium containing 50 ng/ml NGF. NGF is added every two daysand cells were harvested after 4-5 days.

Rat 208F cells were maintained in DMEM plus 10% calf serum in anatmosphere containing 5% CO₂. To generate quiescent cells, the cellswere washed twice with PBS and subsequently grown in DMEM with 0.1% calfserum for 48 hours.

To measure G1 length in Rat-1 cells, the cells were synchronized inpseudometaphase by the addition of nocodazole at 100 ng/ml for 4 hours.The mitotic cells were collected by gentle pipetting. Cells were thenrinsed with DMEM and plated at 2×10⁴ /35 mm dish with DMEM plus 10%bovine calf serum. Cells were pulsed labelled with tritiated thymidine(80 Ci/mmole; 2 μCi/ml) for 30 minutes at each time point. Incorporationof thymidine into DNA was measured as described (Roberts & D'Urso,1988).

Rat-1 cells that constitutively expressed cyclin E were produced asdescribed (Miller and Rosman, 1989). PA317 amphotropic retroviruspackaging cells were plated at 5×10⁵ cells per 60 mm dish on day 1. Onday 2, 1 μg of LXSN-cyclin E, or the control DNA LXSN, was transfectedinto cells using a modification of the calcium phosphate procedure(Ohtsubo et al., 1991). On day 3, the culture medium was replaced withfresh medium and PE501 ecotropic packaging cells were plated 10⁵ cellsper 60 mm dish. On day 4, PE501 cells were fed with 4 ml of fresh mediumcontaining polybrene. Virus was harvested from the PA317 cells and 5 μlto 1 ml of this material were used to infest PE501 cells. On day 5 thePE501 cells were trypsinized and plated in 10 cm dishes in mediumcontaining 0.8 mg/ml G-418. Dishes with small numbers of colonies wereused for isolation of individual clones by using cloning rings. Theseclonal lines were then analyzed by Southern blot analysis and assayedfor vector tiler and suitable clonal lines containing unrearrangedretroviral genomes propagated as virus-producing cell lines. The LXSNand LXSN-cyclin E viruses were used to infect Rat-1 cells and G-418resistant cell populations used for further studies.

Preparation of GST and GST-E columns

E. coli containing the plasmids pGEX-2T or pGEX-2TcycE (GEN-cyclin E)were grown to OD₆₀₀ =0.4 and induced with 0.4 mM IPTG for 4 h at 30° C.Cells were harvested and washed once in PBS and stored at -70° C. GSTencoded by pGEX-2T was prepared as described previously (Koff et al.,1991). Fusion protein GT-cyclin E (GT-cycE) encoded by pGEX-2TcycE wasprepared using a modification of the method of Glotzer et al. (1991).The cell pellet from a 500 ml culture was sonicated in 7 ml of 10mMTris-HCl pH7.4, 0.1M NaCl, 1 mM MgCl₂, 5 mM DTT with proteaseinhibitors. The extract was clarified by centrifugation at 13,000×g andthe supernatant discarded. The pellet was resuspended in 7 ml TND buffer(0.2M Tris-HCl pH 8.2, 0.5M NaCl, 5 mM DTT) and pelleted again. Afterdiscarding the supernatant the pellet was resuspended in 8M ureacontaining 5 mM DTT and mixed gently at 4° C. for 4 hours. The resultingextract was clarified at 13,000×g for 10 minutes and the supernatantdialyzed against TN buffer (i.e., TND buffer containing 1 mM DTT insteadof 5 mM DTT).

At least 2.5 mg of either GT or GT-cycE were incubated with 1 ml ofglutathione agarose beads for 2 hours at 4° C., and subsequentlycollected at 1000×g and washed 3 times with TN buffer containing 1 mMDTT. Coupling of the GT or GT fusion protein to the glutathione agarosesupport was carried out using the following protocol. The support wastransferred to a column and washed with 0.1M borate buffer pH 8.0followed by 0.2M triethanolamine pH 8.2. Dimethylpimelimidate (DMP)cross linker (40 mM DMP, 0.2M triethanolamine pH 8.2) was run into thecolumn leaving just a meniscus. Coupling was continued for 1 hour atroom temperature. After coupling, the column was moved to 4° C. andwashed with 40 mM ethanolamine pH 8.2, followed by 0.1M borate buffer pH8.0. To elute uncoupled protein, the column was washed with PBScontaining 20 mM glutathione pH 7.5 and subsequently stored in PBScontaining 0.5% azide.

H1 kinase assays

8.3×10⁶ cells were lysed by sonication in 100 μl of H1 lysis buffer (50mM Tris-HCl pH 7.4, 0.25M NaCl, 0.5% NP40) containing proteaseinhibitors (1 mM PMSF, 20 μg/ml TPCK, 20 μg/ml SBTI, 10 μg/mlleupeptin). Sonicated lysates were clarified at 13,000×g for 10 minutesat 4° C. and the supernatant transferred to a fresh tube and dilutedtwo-fold with fresh H1 lysis buffer.

50 μl of extract was immunoprecipitated with a 2 μl of polyclonalantisera against cyclin E, or the C-terminus of p34 CDC2 for 1 hour. Forcyclin A immunoprecipitations, lysates were incubated with 5 μl of theC160 monoclonal antibody for 30 minutes and for an additional 30 minutesafter addition of 2 μl of rabbit anti-mouse antibody. Immune complexeswere collected on Protein A sepharose, washed 2× with lysis buffer and4× with H1 kinase buffer (20 mM Tris-HCl pH 7.4, 7.5 mM MgCl₂, 1 mMDTT). H1 kinase reactions were performed as described previously (Koffet al., 1991).

Preparation of lysates for immunoprecipitation-Western blot analysis

Cells (8.3×10⁶ /100 μl) were lysed by sonication in SDS-RIPA (1%deoxycholate, 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl pH 8.0, 0.3MNaCl, 0.1 mM orthovanadate, 50 mM NaF) containing protease inhibitors.In these experiments, approximately 1 mg of affinity purified antibody,or 1 ml of cyclin E pre-immune sera was coupled to 1 ml ofCNBr-activated sepharose according to the manufacturer'srecommendations. In the experiments using cells arrested at the G1/Sboundary, immunoprecipitations were carried out with affinity purifiedantibodies coupled to CNBr-activated sepharose using 2.5×10⁷ cells and100 μl of antibody linked sepharose. For studies of cell cycle fractionsobtained by centrifugal elutriation we used 1×10⁷ cells with 30 μl ofanti-cyclin E sepharose.

Immune complexes were allowed to form for 3 hours at 4° C. and were thenwashed twice with SDS-RIPA containing 5 mg/ml BSA and 3 times withSDS-RIPA. Samples were suspended in Laemmli sample buffer and separatedon 12% PAGE gels. Gels were transferred to nitrocellulose by semi-dryelectroblotting and the membranes blocked with either 2% milk in TNT (25mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) for CDC2 or CDK2, or 1%gelatin in TNT for cyclin E. Blots were probed overnight at roomtemperature with either a 1:300 dilution of affinity purified anti-CDC2,or 1:1000 dilution of anti CDK2 serum, or a 1:1000 dilution of affinitypurified cyclin E antibody. Bound antibody was subsequently detectedwith ¹²⁵ I-Protein A.

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While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1692 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (A) DESCRIPTION: cyclin E cDNA sequence.                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (F) TISSUE TYPE: Human glioblastoma                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: HU4                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 58..1242                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTGCTCACCCGGCCCGGTGCCACCCGGGTCCACAGGGATGCGAAGGAGCGGGACACC57                   ATGAAGGAGGACGGCGGCGCGGAGTTCTCGGCTCGCTCCAGGAAGAGG105                           MetLysGluAspGlyGlyAlaGluPheSerAlaArgSerArgLysArg                              151015                                                                        AAGGCAAACGTGACCGTTTTTTTGCAGGATCCAGATGAAGAAATGGCC153                           LysAlaAsnValThrValPheLeuGlnAspProAspGluGluMetAla                              202530                                                                        AAAATCGACAGGACGGCGAGGGACCAGTGTGGGAGCCAGCCTTGGGAC201                           LysIleAspArgThrAlaArgAspGlnCysGlySerGlnProTrpAsp                              354045                                                                        AATAATGCAGTCTGTGCAGACCCCTGCTCCCTGATCCCCACACCTGAC249                           AsnAsnAlaValCysAlaAspProCysSerLeuIleProThrProAsp                              505560                                                                        AAAGAAGATGATGACCGGGTTTACCCAAACTCAACGTGCAAGCCTCGG297                           LysGluAspAspAspArgValTyrProAsnSerThrCysLysProArg                              65707580                                                                      ATTATTGCACCATCCAGAGGCTCCCCGCTGCCTGTACTGAGCTGGGCA345                           IleIleAlaProSerArgGlySerProLeuProValLeuSerTrpAla                              859095                                                                        AATAGAGAGGAAGTCTGGAAAATCATGTTAAACAAGGAAAAGACATAC393                           AsnArgGluGluValTrpLysIleMetLeuAsnLysGluLysThrTyr                              100105110                                                                     TTAAGGGATCAGCACTTTCTTGAGCAACACCCTCTTCTGCAGCCAAAA441                           LeuArgAspGlnHisPheLeuGluGlnHisProLeuLeuGlnProLys                              115120125                                                                     ATGCGAGCAATTCTTCTGGATTGGTTAATGGAGGTGTGTGAAGTCTAT489                           MetArgAlaIleLeuLeuAspTrpLeuMetGluValCysGluValTyr                              130135140                                                                     AAACTTCACAGGGAGACCTTTTACTTGGCACAAGATTTCTTTGACCGG537                           LysLeuHisArgGluThrPheTyrLeuAlaGlnAspPhePheAspArg                              145150155160                                                                  TATATGGCGACACAAGAAAATGTTGTAAAAACTCTTTTACAGCTTATT585                           TyrMetAlaThrGlnGluAsnValValLysThrLeuLeuGlnLeuIle                              165170175                                                                     GGGATTTCATCTTTATTTATTGCAGCCAAACTTGAGGAAATCTATCCT633                           GlyIleSerSerLeuPheIleAlaAlaLysLeuGluGluIleTyrPro                              180185190                                                                     CCAAAGTTGCACCAGTTTGCGTATGTGACAGATGGAGCTTGTTCAGGA681                           ProLysLeuHisGlnPheAlaTyrValThrAspGlyAlaCysSerGly                              195200205                                                                     GATGAAATTCTCACCATGGAATTAATGATTATGAAGGCCCTTAAGTGG729                           AspGluIleLeuThrMetGluLeuMetIleMetLysAlaLeuLysTrp                              210215220                                                                     CGTTTAAGTCCCCTGACTATTGTGTCCTGGCTGAATGTATACATGCAG777                           ArgLeuSerProLeuThrIleValSerTrpLeuAsnValTyrMetGln                              225230235240                                                                  GTTGCATATCTAAATGACTTACATGAAGTGCTACTGCCGCAGTATCCC825                           ValAlaTyrLeuAsnAspLeuHisGluValLeuLeuProGlnTyrPro                              245250255                                                                     CAGCAAATCTTTATACAGATTGCAGAGCTGTTGGATCTCTGTGTCCTG873                           GlnGlnIlePheIleGlnIleAlaGluLeuLeuAspLeuCysValLeu                              260265270                                                                     GATGTTGACTGCCTTGAATTTCCTTATGGTATACTTGCTGCTTCGGCC921                           AspValAspCysLeuGluPheProTyrGlyIleLeuAlaAlaSerAla                              275280285                                                                     TTGTATCATTTCTCGTCATCTGAATTGATGCAAAAGGTTTCAGGGTAT969                           LeuTyrHisPheSerSerSerGluLeuMetGlnLysValSerGlyTyr                              290295300                                                                     CAGTGGTGCGACATAGAGAACTGTGTCAAGTGGATGGTTCCATTTGCC1017                          GlnTrpCysAspIleGluAsnCysValLysTrpMetValProPheAla                              305310315320                                                                  ATGGTTATAAGGGAGACGGGGAGCTCAAAACTGAAGCACTTCAGGGGC1065                          MetValIleArgGluThrGlySerSerLysLeuLysHisPheArgGly                              325330335                                                                     GTCGCTGATGAAGATGCACACAACATACAGACCCACAGAGACAGCTTG1113                          ValAlaAspGluAspAlaHisAsnIleGlnThrHisArgAspSerLeu                              340345350                                                                     GATTTGCTGGACAAAGCCCGAGCAAAGAAAGCCATGTTGTCTGAACAA1161                          AspLeuLeuAspLysAlaArgAlaLysLysAlaMetLeuSerGluGln                              355360365                                                                     AATAGGGCTTCTCCTCTCCCCAGTGGGCTCCTCACCCCGCCACAGAGC1209                          AsnArgAlaSerProLeuProSerGlyLeuLeuThrProProGlnSer                              370375380                                                                     GGTAAGAAGCAGAGCAGCGGGCCGGAAATGGCGTGACCACCCCATCCTTCTCC1262                     GlyLysLysGlnSerSerGlyProGluMetAla                                             385390395                                                                     ACCAAAGACAGTTGCGCGCCTGCTCCACGTTCTCTTCTGTCTGTTGCAGCGGAGGCGTGC1322              GTTTGCTTTTACAGATATCTGAATGGAAGAGTGTTTCTTCCACAACAGAAGTATTTCTGT1382              GGATGGCATCAAACAGGGCAAAGTGTTTTTTATTGAATGCTTATAGGTTTTTTTTAAATA1442              AGTGGGTCAAGTACACCAGCCACCTCCAGACACCAGTGCGTGCTCCCGATGCTGCTATGG1502              AAGGTGCTACTTGACCTAAAGGACTCCCACAACAACAAAAGCTTGAAGCTGTGGAGGGCC1562              ACGGTGGCGTGGCTCTCCTCGCAGGTGTTCTGGGCTCCGTTGTACCAAGTGGAGCAGGTG1622              GTTGCGGGCAAGCGTTGTGCAGAGCCCATAGCCAGCTGGGCAGGGGGCTGCCCTCTCCGC1682              GGCCGCGCGC1692                                                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 395 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (A) DESCRIPTION: cyclin E amino acid sequence.                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLysGluAspGlyGlyAlaGluPheSerAlaArgSerArgLysArg                              151015                                                                        LysAlaAsnValThrValPheLeuGlnAspProAspGluGluMetAla                              202530                                                                        LysIleAspArgThrAlaArgAspGlnCysGlySerGlnProTrpAsp                              354045                                                                        AsnAsnAlaValCysAlaAspProCysSerLeuIleProThrProAsp                              505560                                                                        LysGluAspAspAspArgValTyrProAsnSerThrCysLysProArg                              65707580                                                                      IleIleAlaProSerArgGlySerProLeuProValLeuSerTrpAla                              859095                                                                        AsnArgGluGluValTrpLysIleMetLeuAsnLysGluLysThrTyr                              100105110                                                                     LeuArgAspGlnHisPheLeuGluGlnHisProLeuLeuGlnProLys                              115120125                                                                     MetArgAlaIleLeuLeuAspTrpLeuMetGluValCysGluValTyr                              130135140                                                                     LysLeuHisArgGluThrPheTyrLeuAlaGlnAspPhePheAspArg                              145150155160                                                                  TyrMetAlaThrGlnGluAsnValValLysThrLeuLeuGlnLeuIle                              165170175                                                                     GlyIleSerSerLeuPheIleAlaAlaLysLeuGluGluIleTyrPro                              180185190                                                                     ProLysLeuHisGlnPheAlaTyrValThrAspGlyAlaCysSerGly                              195200205                                                                     AspGluIleLeuThrMetGluLeuMetIleMetLysAlaLeuLysTrp                              210215220                                                                     ArgLeuSerProLeuThrIleValSerTrpLeuAsnValTyrMetGln                              225230235240                                                                  ValAlaTyrLeuAsnAspLeuHisGluValLeuLeuProGlnTyrPro                              245250255                                                                     GlnGlnIlePheIleGlnIleAlaGluLeuLeuAspLeuCysValLeu                              260265270                                                                     AspValAspCysLeuGluPheProTyrGlyIleLeuAlaAlaSerAla                              275280285                                                                     LeuTyrHisPheSerSerSerGluLeuMetGlnLysValSerGlyTyr                              290295300                                                                     GlnTrpCysAspIleGluAsnCysValLysTrpMetValProPheAla                              305310315320                                                                  MetValIleArgGluThrGlySerSerLysLeuLysHisPheArgGly                              325330335                                                                     ValAlaAspGluAspAlaHisAsnIleGlnThrHisArgAspSerLeu                              340345350                                                                     AspLeuLeuAspLysAlaArgAlaLysLysAlaMetLeuSerGluGln                              355360365                                                                     AsnArgAlaSerProLeuProSerGlyLeuLeuThrProProGlnSer                              370375380                                                                     GlyLysLysGlnSerSerGlyProGluMetAla                                             385390395                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (A) DESCRIPTION: peptide corresponding to C-terminus of                       human CDC2.                                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: C-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TyrLeuAspAsnGlnIleLysLysMet                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (A) DESCRIPTION: peptide from conserved "PSTAIRE"domain of                    CDC2.                                                                         (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CysGluGlyValProSerThrAlaIleArgGluIleSerLeuLeuLys                              151015                                                                        Glu                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (A) DESCRIPTION: peptide corresponding to residues 104-123 of                 human cyclin A.                                                               (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TyrAspGluAlaGluLysGluAlaGlnLysLysProAlaGluSerGln                              151015                                                                        LysIleGluArgGlu                                                               20                                                                            __________________________________________________________________________

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A screening assay foridentifying a compound that modulates or alters cyclin E activity in acell, comprising the steps:establishing replicate test and controlcultures of cells that express cyclin E, administering a candidatecompound to the cells in the test culture but not the control culture,measuring the G1 phase of cells in the test and the control cultures,and determining that the candidate compound modulates or alters cyclin Eactivity in a cell if the G1 phase measured for the test culture isshorter or longer than the G1 phase measured for the control culture. 2.A screening assay for identifying a compound that modulates or alterscyclin E activity in a cell-free system, comprising thesteps:establishing a control system comprising a cyclin E and a celldivision kinase wherein the cyclin E is capable of binding to andactivating the kinase, establishing a test system comprising the cyclinE, the kinase, and a candidate compound, measuring the binding affinityof the cyclin E and the kinase in the control and the test systems, anddetermining that the candidate compound modulates or alters cyclin Eactivity in a cell-free system if the binding affinity measured for thetest system is less than or greater than the binding affinity measuredfor the control system.
 3. A screening assay for identifying a compoundthat modulates or alters cyclin E activity, comprising thesteps:establishing a control system comprising a cyclin E and a celldivision kinase capable of binding cyclin E, establishing a test systemcomprising the cyclin E, the kinase, and a candidate compound, measuringthe activity of cyclin E in the control and the test systems, anddetermining that the candidate compound modulates or alters cyclin Eactivity if the activity of cyclin E in the test system is less than orgreater than the activity measured for the control system.
 4. An assayof claim 3 wherein the control system and the test system areestablished in a cell-free system.
 5. An assay of claim 3 wherein thecontrol system and the test system are established in whole cells.
 6. Anassay of claim 4 wherein the step of measuring comprises measuring thekinase activity in said test and control systems.
 7. An assay of claim 3wherein the step of measuring comprises measuring the binding affinityof the cyclin E and the kinase in the control and the test systems. 8.An assay of claim 3 wherein the step of measuring comprises measuringthe G1 phase of cells in the test and the control cultures.